46th National Congress of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC – Laboratory Medicine)

ALGORITHMS IN ANEMIA DIAGNOSIS

S. Buoro

A. O. Papa Giovanni XXIIII, Bergamo, Italy

Anemia at the threshold of the Third Millennium is a very common pathological condition.

A recent survey conducted by the World Health Organization (WHO) shows that Prevalence among preschool children from industrialized countries varies between 5 to 19%, with the notable exception of United States where is lower than 5%, while in developing countries is never below 19% with nations where Prevalence indexes are generally described above 40%. Things don’t change for the better in women of childbearing age or during pregnancy: Prevalence index is never less than 5% indeed.

Very briefly, Anemia is caused by a defective production and/or increased destruction of erythrocytes, by altered hemoglobin synthesis (impaired or abnormal), by indirect causes like chronic illness or systemic therapy and by acute/chronic bleeding.

In 2002, WHO identifies iron-deficiency anemia as one of the most common cause of illness (1).

Nowadays, there are plenty of algorithm for differential diagnosis of anemia in literature. Such algorithms are based mainly on hemoglobin concentration (Hb) and Wintrobe indices: mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell Hb (MCHC). Althought well-established through their use, these algorithms are no longer able to adequately meet the needs of modern clinical practice and should be supported by other decision-making parameters (2).

Therefore, in differential diagnosis between e.g. iron-deficiency anemia and thalassemia or anemia of chronic disease, traditional parameters of the CBC count are necessary but should include also: reticulocyte count (RET), determination of RET indices (maturation classes and RET hemoglobin content [CHr or Ret-He]), biochemical parameters like serum ferritin, transferrin saturation and finally search for hemoglobin variants in case of suspected hemoglobinopathy.

Besides these so-called ‘Traditional Diagnostic tools’, we are facing a new diagnostic approach based on recent advances in the fields of iron metabolism and homeostasis, with the contribute of peptides and proteins involved in fine regulation of the process: hepcidin, hemojuvelin, HFE, TFR2 genes and Ferroportin. There is a need to introduce therefore new tests based e.g. on genetic and mutational profiles or to strengthen their use, as for Hepcidin dosage (2-3).

Diagnosis of hemolytic anemia should include, in addition to CBC to define type and severity of anemia, any tests that can be able to recognize by definition the hemolitic nature of it (RET, bilirubin, LDH, haptoglobin), combined with ‘etiological’ tests (e.g. Coombs test, search for hemoglobin variants, erythrocyte morphology analysis, serum G6PD and PK levels, analysis of erythrocyte membrane proteins), always from the perspective of the multiparametric approach proposed in the diagnostic algorithm (4).

Similarly, the differential diagnosis of megaloblastic anemia requires the exclusion of Vitamin B12 /Folate deficiency (5). Altough classification schemes for anemia based on the only CBC parameters are necessary, they do not fulfill the need of a complete overview of the anemic status and especially do not reflect/express/represent the current clinical state-of- the-art.

References

1. Worldwide prevalence of anaemia 1993–2005: WHO global database on anaemia / Edited by Bruno de Benoist, Erin McLean, Ines Egli and Mary Cogswell. ISBN 978 92 4 159665 7 (NLM classification: WH 155).

2. Brugnara C, Mohandas N. Red cell indices in classification and treatment of anemias: from M.M. Wintrobes’s original 1934 classification to the third millennium. Curr Opin Hematol. 2013 May;20(3):222-30.

3. Camaschella C, Poggiali E Inherited disorders of iron metabolism. Curr Opin Pediatr. 2011 Feb;23(1):14-20.

4. Dhaliwal G., Cornett P, Tierney LM Hemolytic Anemia Am Fam Physician 2004;69:2599-2606.

5. Kaferle J1, Strzoda CE. Evaluation of macrocytosis. Am Fam Physician. 2009 Feb 1;79(3):203-8.

THE RETICULOCYTES

M. Buttarello

Dipartimento dei Servizi di Diagnosi e Cura, Azienda ULSS 19, Adria, Rovigo, Italy

In 1995, Martin Rowan, late lamented Secretary of the expert panel on cytometry of ICSH, wrote an editorial titled “reticulocyte counting: resurrection and respectability.”

The title emphasized an effective change about the possibility to use the reticulocyte count more extensively than in the past and, at the same time, the ability to obtain more information on the reticulocyte cell as the fraction of immature reticulocytes (IRF), with new clinical applications.

In fact, during the years’ 80-90 flow cytometry entered with force in the reticulocyte counting replacing the traditional microscopic method by use of two key elements: 1) the ability to analyze tens of thousands of cells per sample and thus reduce the statistical error affecting inevitably the manual method (and therefore reduce the imprecision); 2) the availability of commercial dyes (fluorescent and not) capable of binding to ribosomal RNA of reticulocytes and then to allow a count of the cells containing RNA, overcoming the one hand the error connected to the subjectivity of interpretation, and on the other providing a quantitative measure proportional to the amount of RNA and therefore the level of maturation of the reticulocyte.

Later, with some automated systems, it was also possible to measure the mean hemoglobin content (CHr or Ret-He) and the mean volume of reticulocytes (MCVr), also known as reticolocyte indices, similarly to the corresponding red cell indices, providing additional diagnostic applications.

The combination of precise and accurate count with the IRF value has proved useful in distinguishing anemias characterized by increased bone marrow erythropoiesis (increase in reticulocyte and in the IRF), from anemias due to reduced marrow activity (reduction of both), and from situations in which there is a dissociation between the reticulocyte count (low or normal) and IRF (increased) as in myelodysplastic syndromes and in acute infections. In reticulocytopenic patients undergoing bone marrow transplantation or chemotherapy, an increase IRF is the earliest sign of marrow regeneration.

Of the reticulocyte indices the most promising are the CHr which directly reflects the synthesis of hemoglobin in marrow precursors and is therefore a measure of the adequacy of iron availability: its reduction indicates iron- deficient erythropoiesis. This index has proved useful for monitoring early response to intravenous iron administration (with significant increases after only 48 hours). Low values of CHr are indicative of iron-deficient erythropoiesis in patients undergoing dialysis and even in functional iron deficiency as in the treatment with erythropoietin. In subjects with depleted iron stores the MCVr increases rapidly following iron therapy and decreases equally as rapidly with the development of iron deficient erythropoiesis. MCVr decreases and reticulocytes are smaller than the circulating red blood cells in macrocytosis after therapy with B12 and/or folate. Therefore CHr and MCVr have many overlapping clinical applications.

References

1. Rowan R.M. Reticulocyte Counting: Resurrection and Respectability. Sysmex J Int 1995; 5: 74-76.

2. Buttarello M, Bulian P, Farina G, et al. Flow cytometric reticulocyte counting. Parallel evaluation of five fully automated analyzers: an NCCLS-ICSH approach. Am J Clin Pathol 2001; 115:100-111.

3. Buttarello M, Bulian P, Farina G, et al. Five fully automated methods for performing immature reticulocyte fraction. Comparison in diagnosis of bone marrow aplasia. Am J Clin Pathol 2002; 117: 871- 879.

4. Buttarello M, Plebani M. Automated blood cell counts. State of the arts. Am J Clin Pathol 2008; 130: 104-116.

5. Piva E, Brugnara C, Chiandetti L, Plebani M. Automated reticulocyte counting: state of the art and clinical applications in the evaluation of erythropoiesis. Clin Chem Lab Med 2010; 48: 1369-1380.

A CASE OF REFRACTORY CYTOPENIA WITH MULTILINEAGE DYSPLASIA ASSOCIATED WITH BONE MARROW FIBROSIS

F. Fiorini¹, M. Bombara², A. La Gioia¹

¹U.O. Patologia Clinica, Osp. “F. Lotti” Pontedera, Pisa

²Laboratorio di Analisi Chimico-Cliniche e Microbiologia Spedali Riuniti, Livorno

The myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell characterized by peripheral cytopenia and dysplasia associated with dysplasia and hyperplasia on the bone marrow. The progression to acute myeloid leukemia is frequent.

Actually, the 2008 World Health Organization (WHO) classification, provides the best diagnostic approach to MDS and it is also important for prognostic evaluations (1).

The most important criteria to classify MDS are the thresholds for different types of cytopenia, the blast cells count and the dysplasia. The morphological features of dysplasia can affect the red cell, granulocytic and platelet’s lines. However, on the WHO classification, there are cases that do not meet these criteria: idiopathic cytopenia of uncertain significance and idiopathic dysplasia of uncertain significance (cytopenia without dysplasia and, respectively, dysplasia without cytopenia); the hypoplastic myelodysplastic syndrome; the myelodysplastic syndrome with fibrosis (MDS-F).

We describe a case of MDS, with prominent, unusual dysplastic features and myelofibrosis. An 84-year-old woman presented with bi-cytopenia (Hemoglobin, 7.0 g/ dL; neutrophils, 0.6 x 10⁹/L). In her bone marrow, the majority of the nucleated cells (80%) were erythroblasts.

Blasts were up to 4% of the nucleated cells (21% of the non-erythroid cells) without Auer rods. Granulocytic cells were 15% and lymphocytes 1%. Dyserythropoiesis and dysmegakaryocytosis were prominent including usual features (megaloblastic changes, multi-nuclearity, internuclear bridging and ringed sideroblasts) and unusual features (erythrophagocytosis, hot spots, asynchronous mitosis). The silver stained sections revealed a grade 2 fibrosis on 0- 3 scale, according to European Consensus Scoring Systems: 0-3 score.

The patient was diagnosed as refractory cytopenia with multilineage dysplasia (RCMD) associated with fibrosis. The current WHO classification neither provides a suitable diagnostic category for these cases nor indicates these unusual features as dyserythropoiesis. RCMD with fibrosis might be a new provisional category in MDS.

Reference

1. Brunning RD, Orazi A, Germing U, et al. Myelodysplastic syndromes/neoplasms, overview. In: Swerdlow SH, Campo E, Lee Harris N, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC, 2008:88-93.

APPLICATION OF THERMOGRAVIMETRY AND CHEMOMETRICS FOR β-THALASSEMIA CHARACTERIZATION

R. Risoluti¹, S. Materazzi¹, F. Sorrentino², P. Caprari³

¹Department of Chemistry, “Sapienza”, University of Rome, Rome

²DH-Thalassemia, S. Eugenio Hospital, Rome³Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome

β-Thalassemia is one of most common autosomal recessive disorders worldwide. β-Thalassemia is caused by the reduced or absent synthesis of the β globin chains of the hemoglobin tetramer. Three clinical and hematological conditions of increasing severity are recognized, i.e. the β-thalassemia carrier state, β-thalassemia intermedia, and β- thalassemia major. The hematological features are microcytosis, hypochromia, and anemia. In this study the possibility of using thermogravimetric analysis (TGA) followed by chemometrics as a new approach for β-thalassemia detection is proposed. Blood samples from patients with β-thalassemia were analyzed by the TG7 thermobalance (Perkin Elmer) and the resulting curves were compared to those typical of healthy individuals. In order to characterize the severity of anemia, the hematological parameters were determined by ADVIA 120 analyzer (Siemens). The thermogravimetric profiles of blood samples from β-thalassemic patients were clearly distinct from those of healthy individuals as result of the different amounts of water content and corpuscular fraction. The hematological overview showed significant decrease in Hb, Hct, MCH, and MCV values and increase in RBC counts and RDW values in thalassemic subjects as compared with healthy subjects. Principal Component Analysis (PCA) was used to evaluate the correlation between the haematological parameters, characteristic of β-thalassemia, and the thermogravimetric results. The implementation of a predictive classification model allows to consider thermogravimetry followed by chemometrics as a fast, rapid and cost-effective diagnostic tool for βthalassemia detection.

A CASE OF ATYPICAL URTICARIA

M. Marinova¹, M. Nabergoj², C. Artusi¹, S. Baggio¹, G. Antonelli³, L. Brugnolo¹, M. Zaninotto¹, M. Plebani^1,3

¹Department of Laboratory Medicine, University-Hospital, Padova, Italy

²Hematology and Clinical Immunology, University-Hospital, Padova, Italy

³Department of Medicine, University of Padova, Italy

A 50-year-old woman was admitted to the hospital for high remittent fever associated with widespread pruritic maculopapular erythema and sore throat starting a week prior to her admission. In the last two days she complained of asthenia and diffuse muscle pain with acute exacerbation in close relationship with fever spikes.

Examination revealed a well built female with fever of 38.3°C, latero-cervical lymphadenopathy, hepatomegaly and highly congested throat. Numerous confluent, erythematous, urticarial macules and papules that coalesced into urticarial plaques were observed.

Hematological investigations showed leucocytosis of 20.8 x 10⁹/L (87.0% neutrophils). Both the acute phase reactants were high, with C-reactive protein of 300 mg/L and erythrocyte sedimentation rate of 62 mm/hr. There were markedly elevated levels of serum ferritin (20.900 ug/L) and mild liver dysfunction (AST: 44 U/L, ALT: 62 U/L). All other hemato-biochemical and serological tests were within the normal ranges. Blood and urine cultures revealed no evidence of bacterial, fungal or viral infection. A glycosylated ferritin assay was performed due to the extreme hyperferritinaemia, showing a reduction of the glycosylated fraction (<20%), a newly proposed diagnostic criteria for Adult Still’s disease (AOSD).

AOSD is a rare systematic inflammatory disorder with unknown etiology and pathogenesis (1/100000). The main features are: prolonged fever, arthritis, evanescent nonpruritic, macular, and salmon coloured rash, pharyngitis, and leucocytosis with neutrophilia. Organomegaly, lymphadenopathy, myalgia, serositis, and septic meningitis can also occur. The clinical manifestation of this disease could mimic an infectious syndrome, malignancies and rheumatic diseases. Thus, AOSD is still an exclusion diagnosis. The authors report a case of AOSD with atypical skin rash. We should emphasize the great diagnostic value of glycosylated ferritin for the correct diagnosis, an assay rarely available in a routine laboratory.

Initially, antibiotic and low dosage corticosteroid therapy was introduced, without any improvement. After the diagnosis of AOSD, administration of high-dose corticosteroids and Cyclosporin A dramatically improved the patient’s clinical and biochemical status with no recurrence.

A CASE OF MONOCLONAL GAMMOPATHY OF RENAL SIGNIFICANCE IN A PATIENT AFFECTED BY IMMUNOTACTOID GLOMERULOPATHY

A. Terreni¹, F. Bergesio², E. Dervishi², C. Nozzoli³, E. Buti², K. Giannakakis⁴, M. Brogi¹, T. Biagioli¹, E. Minetti², L. Cirami², A. Caldini¹

¹Lab. Generale, AOU-Careggi, Firenze

²Nefrologia e Dialisi, AOU-Careggi, Firenze

³Ematologia, AOU-Careggi, Firenze

⁴Dip. di Patologia, Università La Sapienza, Roma

The term Monoclonal Gammopathy of Renal Significance (MGRS) indicates a causal relationship between monoclonal gammopathy (MG) and kidney disease (KD) in patients in which the significance of MG is not undetermined, but it is linked to the pathogenesis of the disease. We report a case of MGRS, in a patient with immunotactoid glomerulopathy (ITG), a rare disease characterized by glomerular deposition of Congo Red negative microtubules.

A man (58aa) with history of hypertension, dyslipidemia, obesity, non-nephrotic proteinuria (NNP) (2gr/24h) came to our attention for a suspected cryoglobulinemic disease.

Laboratory findings showed a monoclonal component (MC) IgGk (7 g/L), free light chains (FLCs) slightly altered (k=44.7 mg/L, λ=22.9 mg/L) and negative Bence Jones (BJ). No evidence of autoimmune or cryoglobulinemic disease was present. A renal biopsy was performed, but the diagnosis remained uncertain between a form of occult cryoglobulinemic glomerulopathy and ITG. Bone marrow biopsy (BOM) was not performed. The patient was discharged and monitored for renal function and cryoglobulinemia, the last of which, despite all our efforts, never yielded a positive result. After 3 years, the patient was hospitalized due to KD (sCr=1.6 mg/dL, CrCl=45 mL/min) and for persistence of NNP. A second renal biopsy was performed: light microscopy revealed mesangial and sub-endothelial deposits which on immunofluorescence stained for IgG, IgM, C3 and kappa light chains. Electron microscopy revealed microtubules of about 20 nm in thickness. The MC was 9.3 g/L, with FLCs k=25.6 mg/ L, λ=12 mg/L, and BJ was negative. The BOM showed 40% plasmacells producing IgG monotypic for k light chain. The MC IgGK was very likely to be responsible for renal deposits. An ITG was diagnosed and the patient was treated with bortezomib and dexamethasone. After 5 cycles the MC was 7.6 g/L, FLC k=8 mg/L and λ=5 mg/L, sCr 1.3 mg/dL, CrCl=83 mL/min and proteinuria reduced by 50 %. The patient died due to complications after autologous BM transplant. MC reduction and KD improvement after treatment, strongly support the MC pathogenetic role. This case highlights the importance of further investigations when a MC, accompanied by KD, is present regardless if small, for its possible renal significance.

PROLONGED ACTIVATED PARTIAL THROMBOPLASTIN TIME IN AN ASYMPTOMATIC PATIENT: THE PREKALLIKREIN DEFICIENCY

L. Tomao², A. Antenucci¹, C. Mandoj¹, L. Autullo¹, G. Vercillo¹, L. Conti¹, M.G. D’Alessandro¹

¹Clinical Pathology, “Regina Elena” National Cancer Institute, IRCCS Rome

²Department of Sciences, University of “Roma Tre”, Rome

Prekallikrein (PK), also known as Fletcher factor, is a contact factor that complexes with high molecular weight kininogen and is cleaved by factor XII to produce kallikrein in the very first part of the intrinsic pathway of the coagulation cascade. Patients with PK deficiency usually do not show a bleeding tendency, so this particular condition is often detected in asymptomatic subjects after an incidental finding of isolated, and apparently inexplicable, prolonged activated Partial Thromboplastin Time (aPTT).

Here we report the case of a 72-years-old Caucasian woman with a history of hypertension and hyperlipidemia who entered the Clinical Pathology Unit of Regina Elena National Cancer Institute. Incidentally, the patient was found to have prolonged aPTT test (137.9 sec) and normal Prothrombin Time (PT) test (108%) and no indication of a concomitant anticoagulant therapy. The correction to normal values of the aPTT test after mixing patient’s plasma with an equal volume of normal pool plasma, indicated a coagulation factor deficiency, but testing factors XII, XI, IX and VIII activities we found always normal values, just like the von Willebrand factor (vWf) antigen quantification.

Following these results, a deficit in the contact phase of the intrinsic coagulation pathway was suspected, more particularly a PK deficiency. Waiting for the PK deficient plasma to arrive, we performed the aPTT test prolonging subsequently the preincubation time of patient’s plasma with the aPTT reagent. This has been done using the Synthasil (IL) reagent that showed the best sensitivity when compared to other reagents (e.g. Synthafax) that underestimated the aPTT prolongation, causing confusion on the patients situation. In this case a normalization of aPTT value was found.

This experience shows that a situation of prolonged aPTT test in absence of bleeding events is not only referable to the development of an acquired thrombophilia syndrome (e.g. Lupus Anticoagulant) but it could be, as in this case, a rare deficiency in the very first part of the coagulation cascade activation.

Reference

1. Acar K, Yağci M, Sucak GT, et al. Isolated prolonged activated partial thromboplastin time in an asymptomatic patient: Fletcher factor deficiency. Thromb Res 2006;118:765-6.

THE ROLE OF THE CA15-3 IN GUIDING THE THERAPY IN METASTATIC BREAST CANCER: A CASE REPORT

C. Cocco¹, B. Caruso¹, E. Fiorio²

¹Lab. Analisi dO, AOUI Verona

²Oncologia Medica dO, AOUI Verona

Background: The National Academy of Clinical Biochemistry states that CA15-3 in combination with imaging and clinical examinations can be used to monitor the therapy in patients with advanced breast cancer. Despite some limitations, like the transient increase of the marker that can occur after the initiation of therapy (also known as surge), its use may be helpful in specific patients, particularly when the imaging techniques are not sensitive enough to demonstrate the progression of the disease.

Case report: A 62 year old patient with a 2 year history of invasive ductal carcinoma of the breast, treated with an Aromatase inhibitor was admitted in March 2012 to the hospital. Computed Tomography (CT) showed bone marrow infiltration, liver and bone metastases. The value of CA 15-3, measured by CentaurXP (Siemens) was high: 540 KU/L (r.i. 5-35 KU/L). The oncologist decided to modify the therapy using a mitotic inhibitor (Taxano). A CT scan was arranged after 3 months and CA15-3 measurements were planned every 21 days. The tumor marker, after an initial rise to 600 KU/L, showed a significant decrease to 150 KU/L. In the following 3 months, the CA15-3 values ranged between 150 and 180 KU/L while the CT scan documented a disease progression in the liver. A monoclonal antibody blocking angiogenesis was then added. During the following 3 months, despite the new drug, the serum concentration of CA15- 3 remained unchanged. The therapy was modified again replacing the mitotic inhibitor with a non steroidal antiestrogen. For 3 weeks the CA15-3 showed a surge (810 KU/L), then a rapid decrease in the range of 150-180 KU/L was observed. Next CT scan was unable to show any modification compared to the previous one. On the basis of the values of the CA15-3, still above the reference range, and on clinical judgment, a dual chemotherapy (Capecitabine and Vinorelbine) was introduced. In the following months a gradual decrease of the marker down to the reference range was observed and the diagnostic imaging showed a partial regression of the disease.

Conclusions: This case report shows that the serum concentrations of the tumor marker have contributed to a large extent to stop ineffective treatments and have effectively helped the clinician in selecting the most appropriate treatment options.

CHROMOSOME 22Q11.21 DUPLICATION IN A PATIENT WITH CONGENITAL HEART DEFECT

C. Munno¹, F. Verdesca¹, A. Vitale², B. Lombardo¹, L. Pastore¹

¹Ceinge-Biotecnologie Avanzate, Dip. di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, Naples, Italy

²Dip. di Scienze Motorie e del Benessere Università di Napoli “Parthenope”, Naples, Italy

Background: The newly described 22q11.2 microduplication syndrome is the association of a broad clinical spectrum and up till now more than 50 unrelated cases have been reported with a high frequency of familial duplications. The clinical presentation of patients is extremely variable (1) and shares features with 22q11.2 deletion syndromes (DG/VCFS), including heart defects (2), urogenital abnormalities, velopharyngeal insufficiency with or without cleft palate, and ranging from multiple defects to mild learning difficulties with some individuals being essentially normal. The basis of this clinical variability remains unclear.

Methods: A high resolution array comparative genomic hybridization (a-CGH) 4x180K was performed on the patient with congenital heart defect, a pulmonary valve stenosis, in order to identify potential mutation and characterize the clinical phenotype at molecular level. The a-CGH used contains 170,334 60-mer oligonucleotide probes that cover the whole genome with an average spatial resolution of 13 Kb. DNA digestion, labeling and hybridization were performed according to the manufacturer’s protocols.

Results and conclusions: Using a-CGH analysis, we identified a duplication in 22q11.22 region of approximately 2,5 Mbp; the aberration observed encompasses a region containing 60 genes including several OMIM genes such as TBX1, PROH, GP1BB, COMT, RTN4R, SCARF2, SERPIND1 e SNAP29. We analyzed by a-CGH a patient with a congenital heart defect and identified a microduplication 22q11.21 region of approximately 2,5 Mbp as potential causative mutation. Is known that the frequency of the duplications in 22q11.21 region is approximately half that of the deletions. This difference might be explained either by the wide range and sometimes mild phenotypes of the patients, which will give less reason to suspect and test for 22q11.2 duplication or to technical difficulties in detecting microduplications by fluorescent in situ hybridisation (FISH) on metaphase spreads. With the clinical implementation of genomic microarrays the number of patients detected with 22q11.2 duplication is almost certainly going to increase with the aim of delineating the phenotype of the 22q11.2 duplication syndrome.

ACUTE MYELOID LEUKEMIA WITH t(11,22)(q23;q13) TRANSLOCATION, A CASE REPORT

G. Pantano, F. Tosato, M.C. Sanzari, E. Piva, P. Fogar, M. Plebani

Department of Laboratory Medicine, University Hospital of Padova

Background: the t(11,22)(q23;q13) translocation, which causes the MLL gene rearrangement, has so far been described in the literature in only 4 cases of acute leukemia.

Methods: 34 years old Caucasian man, admitted in Medicine with pancytopenia and severe acute kidney falure. History: L4-S1 stabilization for congenital spondylolisthesis with the use of blood autotransfusion; since then, moderate recurrent low back pain; 2 recent Emergency Room visits for acute low back pain, that was treated with analgesic and anti-inflammatory drugs. The patient undergoes, in the Hematology Department, a diagnostic bone marrow biopsy and aspirate, in the suspicion of: hemolytic uremic syndrome, Moschcowitz syndrome or acute leukemia.

Results: the morphological examination of the bone marrow smears reveals that the myeloid lineage is mainly represented by likely leukemic promyelocytes characterized by: medium-large size, slightly immature chromatin, occasionally visible nucleoli, large cytoplasm with azurophilic granules, absence of Auer rods. Flow cytometric analysis identifies blasts (65%) with immunophenotype: CD34- CD117+ MPO+ CD33+ CD13+ CD15+ HLA DR- CD11b- CD16- CD56- CD66b- CD64- CD14- CD38+ CD4+, consistent with possible acute promyelocytic leukemia, variant form. Cytogenetic analysis identifies the t(11,22)(q23;q13) balanced translocation in the 100% of the analysed metaphases, no other chromosomal aberrations are found. The translocation causes the MLL gene rearrangement, as confirmed by the FISH. The MLL gene is rearranged in about the 7-8% of the acute myeloid leukemia (AML) with abnormal karyotype. At the beginning, the patient is treated with ATRA, with only partial benefit, then he is treated with the ARA-C + IDA (3+7) chemotherapy protocol, obtaining the remission. Subsequently, the patient undergoes consolidation chemotherapy and bone marrow transplantasion, because of the high risk of relapse.

Conclusion: the t(11,22)(q23;q13) translocation, which leads to the formation of the MLL/EP300 fusion transcript, has so far been identified in only 4 cases: 2 therapy related AML, 1 AML with myelodisplasia related changes, and 1 acute lymphoblastic leukemia. Here, the translocation has been found in an acute leukemia, which could be classified as an acute promyelocytic leukemia according to the morphological and immunophenotypical criteria, but not using the cytological ones.

Reference

1. Novel variant form of t(11;22)(q23;q13)/MLL-EP300 fusion transcript in the evolution of an acute myeloid leukemia with myelodysplasia-related changes. Duhoux FP, De Wilde S, Ameye G, et al. Leukemia Research 2011;35:e18-e20.

GLUTEN HYPERSENSITIVITY: BASOPHIL ACTIVATION TEST (BAT) MAY HELP IN THE DIFFERENTIAL DIAGNOSIS?

A.T. Scacchetti¹, D. Debbia¹, E. Boni², T. Trenti¹

¹Dip. Interaziendale ad Attività Integrata “Medicina di laboratorio e Anatomia Patologica” NOCSAE, Modena

²Dip. di Clinica e Medicina Sperimentale, Università di Parma

Background: The main forms of gluten-related disorders are wheat allergy, autoimmune disease (celiac disease, dermatitis herpetiformis and gluten ataxia) and possibly immune-mediated disease (gluten hypersensitivity).

IgE antibodies play an essential role in the allergic disease. For celiac disease (CD) the available tests are anti-tissue transglutaminase (tTG) IgA, anti-endomysial antibodies (EMA) and deamidated gliadin peptides (DGP) antibodies especially IgG class. For other immune-mediated disorders there is currently no tests available.

There is evidence that the BAT reveal reactions of IgE and non-IgE-mediated. The BAT based on the detection of CRTH2/CD203c in whole blood sample is based on evaluation of membrane molecule (CD63) that is expressed on the cell surface of basophils by flow cytometry, after in vitro antigenic stimulus.

Methods: A 46-years-old woman with wheat-related disorder manifesting clinical signs, intestinal symptoms (chronic diarrhea, weight loss) and extraintestinal symptoms (chronic orticaria-angioedema, fatigue). The following methods were employed: Skin Prick Test (SPT) (Lofarma) in vivo; Specific IgE (S-IgE) (Thermo Fisher Scientific), DGP IgG and IgA anti-tTG Ab (Thermo Fisher Scientific), EMA (Eurospital), HLA (Euroimmune) in vitro. It was also made the BAT (Beckman Coulter) and was used as antigenic stimulus extract grain (Lofarma).

Results: SPT and S-IgE was negative for wheat: f4 =<0.10 KUa/L; F79 (gluten) = <0.10 KUa/L; g2 (Poa pratensis) =<0.10 KUa/L; g8 (Cynodon dactylon) = 0.54 KUa/L; DGP IgG/IgA tTG IgA/IgG, EMA were negative; HLA-DQ2/DQ8 not present; Endoscopy of the digestive tract has been documented in the standard. The BAT (52.9%, c.o.=>15%) showed an activation of basophils.

Conclusions: Shortly after a strict gluten-free diet, the patient’s clinical condition improved.

All the data excluded an allergic or autoimmune disease. The fact that the clinical signs and symptoms completely resolved after the diet, demonstrates the implication of gluten exposure in the pathogenesis. The diagnosis of hypersensitivity to gluten has been paid on the basis of the algorithm for the differential diagnosis of disorders related to gluten (Sapone et al. BMC Med. 2012) The BAT has confirmed a hypersensitivity reaction to wheat (not-IgE-mediated and not CD).

THE CLINICAL RELEVANCE OF NRBC: AN UNUSUAL ONSET OF RAEB-2

M. Moretti¹, F. Centis¹, S. Rapa¹, B. Pieretti¹, M.R. Sadori¹, D. Sbrozzi¹, G. Tombari¹, S. Gasperoni¹

¹Patologia Clinica, AO Ospedali Riuniti Marche Nord (PU)

Background: Nucleated red blood cells (NRBC) are normally absent in adult peripheral blood (PB), their presence is a signal of a severe hematological stress. The new generation of hematology analyzers improved the accuracy in NRBC detection and count (better specificity and sensitivity). Refractory anemia with excess blast (RAEB) is a myelodysplastic syndrome characterized by ineffective hematopoiesis and dyserytropoiesis. Two categories of RAEB are recognized: RAEB-1 defined by 5-9% of blasts in the bone marrow (BM) or 2-4% blasts in the PB and RAEB-2 defined by 10-19% of blasts in the BM or 5-19% blasts in PB. We reported an unusual onset of RAEB-2 with great amount of NRBC in PB.

Case report: A complete blood count was ordered for a 73 year old woman with asthenia, weight loss since two months. Sample analysis was conducted by Sysmex XN (XN) hematology analyzer that counts NRBC utilizing flow cytometry, light and fluorescent scatter. The XN analysis and PB smear optical microscopic evaluation (OM) showed: 5.68X10ˆ9/L white blood cells (WBC), (70% neutrophils, 12% lymphocites, 6% monocytes, 6% blasts, 3% promyelocytes and 3% myelocytes); 82X10ˆ9/L platelets; 4.8 g/dL hemoglobin, 2.07X10ˆ12/L RBC, 108 NRBC/100 WBC. XN correctly flagged the presence of myeloid immature cells, blasts and counted with good agreement versus OM the amount of NRBC despite the presence of several dyserythropoiesis features. Laboratory results rapidly triggered the diagnostic work-up. BM was hypercellular with erythroid hyperplasia, myelo/thrombocytopenia, dysgranulo and dysmegakaryocytopoiesis, alteration of the histotopography with mild increase in reticuline. Blast cells 12%. Immunophenotyping showed increased levels of CD34+/ CD117+ cells (myeloblast 8%). 60% of the interphase nuclei showed del(5q), 7 monosomy, del(12p). Definitive diagnosis was RAEB-2, International Prognostic Scoring System 2. Patient started treatment with azacitidina and today after five months she has mild leuco/thrombocytopenia and anemia that requests sometimes transfusions. During this period analysis by XN and OM have not detected NRBC in the PB.

Conclusion: NRBC in PB and severe anemia are the principal features of that unusual onset of RAEB-2. Accurate automated NRBC count plays a key role to trigger the diagnostic work-up and for the follow up.

A MYELOPATHY OF UNCERTAIN ORIGIN

M.T. Muratore¹, R. Buzzi¹, B. Mongiardo², V. Durastanti³, G. Salvatori³, R. Carrozza¹

¹U.O.C. Laboratorio Analisi, Osp. Belcolle, Viterbo

²U.O.C. Medicina d’Urgenza, Osp. Belcolle, Viterbo

³U.O.C. Neurologia, Osp. Belcolle, Viterbo

In November 2013, after a week-long febrile episode, a 51-year-old male reports first feeling exhausted and then increasing clumsiness and rigidity of the lower limbs. About two months after the onset of the first symptoms, neurological examination shows: ataxic-spastic gait, diffuse accentuation of the deep tendon reflexes with extensor plantar response(more defined on the right side), abolition of the abdominal reflexes. Suspecting an autoimmune para-infectious myelo-neuropathy, a series of investigations are +evaluation of the serum free light chains shows an altered ratio k / λ (3,2). All other examinations of clinical chemistry, hematology (TSH, PSA)and autoimmunity (including antigangliosides antibodies) result normal. The nerve conduction studies show signs of a very mild sensory neuropathy (slight reduction in amplitude of the SEP of the ulnar and sural nerve, normal CMAP from all motor nerves examined). The pattern-reversal visual evoked potential shows bilateral increase of P100 latency (121 msec in the Right eye; 115 msec in the Left). MRI with contrast shows a parenchymal lesion of the spinal cord at C2 level, of about 9 mm in length; spinal cord injury does not assume contrast. Clinical and instrumental examinations require differential diagnosis with possible multiple sclerosis. CSF examination shows only a moderate increase of albumin with mild damage to the barrier function of the brain but without oligoclonal reaction: the isolettrofocusing is of type V (special profile reflection due to the passage of serum monoclonal component to the CSF). The determination of free light chains in the CSF seems to confirm the absence of intrathecal synthesis. Since these evidences do not support the diagnosis of MS they suggest instead a likely diagnosis of Devic’s disease (neuromyelitis optica). Further tests are performed on serum to detect aquaporin-4 antibodies (AQP4-Ab; also termed NMO-IgG) leading to positive results. The role of the laboratory has been fundamental to diagnose a disease formerly considered a variant of the MS which is now recognized as a different illness, which has its own clinical and immunological profile.”

THE CSF BIOMARKERS IN A CASE OF CHRONIC SUBARACHNOID MICRO-BLEEDING AND SUPERFICIAL SIDEROSIS

G. Sancesario¹, T. Schirinzi², L. Anemona³, A. Pisani², O. Porzio¹, G. Sancesario², S. Bernardini¹

¹Dept of Experimental Medicine and Surgery, University Hospital of Roma Tor Vergata, Rome, Italy

²Dept of Systems Medicine, University Hospital of Roma Tor Vergata, Italy

³Dept of Biomedicine and Prevention, University Hospital of Roma Tor Vergata, Rome, Italy

We observed a case of superficial siderosis (SS) of central nervous system caused by a post-traumatic avulsion of the left brachial plexus forty years before. We questioned whether SS was the stabilized effect of a remote post-traumatic bleeding or an evolutive actual process indirectly related to the injury. The patient had a thorough clinical assessment and was subjected to brain and spiny magnetic resonance scan (MRI). Moreover, we performed a full cyto-biochemical analysis of cerebrospinal fluid (CSF) evaluating in particular the CSF-ferritin levels as an indirect iron accumulation index and the presence of erythrocytes and macrophages as an index of recurrence of bleeding. MRI shown iron accumulation signals mainly on the cerebellum and the brain stem, which were unchanged after iron chelator deferiprone treatment. CSF before treatment was clear and colorless, the total amount of proteins was 55.10 mg/dL, 9 white cells/mm3 (WBC), no erythrocytes; the microscopic analysis revealed the presence of few macrophages containing fragments of erythrocytes (siderophages). CSF-ferritin level was 76 ng/mL (normal range <12 ng/mL). After treatment for three months, CSF cyto-biochemical analysis was substantially unvaried while the CSF-ferritin level was 61.7 ng/mL, about 20% less than baseline. WBC counts and liver function tests were normal, as the blood levels of iron and ferritin. Considering that siderophages are seen as early as 1–2 days after hemorrhage and may persist for weeks, their presence in CSF demonstrated the occurrence of a condition of persistent or intermittent bleeding. Measurement of CSF-ferritin as an iron deposition index allowed formulating a precise diagnosis of SS. Treatment with deferiprone improved clinical symptoms and slightly reduced CSF-ferritin concentration, even in presence of unchanged neuroimaging. However we cannot exclude that the lowering of CSF-ferritin could be dependent on a spontaneous fluctuation in iron or ferritin concentration or again on a casual transient interruption of subarachnoid microbleeding. The variation in CSF-ferritin concentration compared to a baseline value allowed a more sensitive and precise evaluation of the efficacy of an iron-chelating treatment than the monitoring of hemosiderin signals on MRI.

OLD AND NEW ADDICTIONS

R. Pacifici

Direttore Reparto Farmacodipendenza Tossicodipendenza e Doping, Istituto Superiore di Sanità, Roma Direttore Osservatorio Fumo, Alcol e Droga, Istituto Superiore di Sanità, Roma

Drug addiction is a chronic relapsing disease, linked to cognition and pleasure reward disturbances. Whereas it is easy to recognize in the traditional drugs of abuse, alcohol and tobacco active principles able in inducing physical and/or psychological dependence, the so-called “new addictions” are insidious and unrecognized. Alongside the traditional drugs, the consumption of so-called “new drugs” is increasingly affirming. Those are molecules synthesized in the laboratory with a booming market almost exclusively handled via the Internet.

With regard to alcohol consumption, the epidemiological picture confirms that the “Mediterranean” consumption, characterized mainly by moderate daily wine use, is becoming less widespread in our Country and affects more and more adults and the elderly, while among youth and young adults occasional consumption out-of meals is spreading. Tobacco smoke remains a health issue of great social impact in our country. Smokers in Italy are 11.3 million, 22% of the population: 6.2 million men (25.4%) and 5.1 million women (18.9%).

Alongside the traditional “addictions” new “behaviours” are adopted similar to real addictions. Individuals who experience these new dependencies often associate pathological behaviors to the consumption of traditional drugs of abuse, alcohol, or tobacco. Among the new dependencies gambling, eating disorders, compulsive use of the Internet can be mentioned. In the DSM-5, pathological gambling (PG) was classified under the section titled “Substance-Related and Addictive Disorders”. In the last years, the problem gamblers (those who played at least 20 times in the last year) fell slightly, from 16.5% in 2008 to 11,6% in 2012.

The “food addiction” comes from the loss of control over normal eating behavior. Social and cultural factors are particularly important in terms of the onset of eating disorders (ED). Interestingly, some surveys detect significant differences between the young people who show an increased risk for problematic eating behavior and their peers. Indeed, among the first higher percentages of those who used during the last year at least once cocaine (4% vs 2%), stimulants (5% vs 2%), drugs not prescribed by a doctor (20% vs. 7%) are observed.

Some authors finally argue that the use of new technologies and Internet are able to modify neuronal circuits and thus the way we think, read, remember and organize information. The literature also investigate psychiatric disorders related to excessive use of the media. Among the young people who spend more than 5 hours / day in Internet the risk of incurring a problematic eating behavior (18% vs. 10% of control) or to consume at least one illegal psychoactive substance (22% vs 14%) is higher, so that cannabis use is defined as “problematic” for 28% of these cyber-internauts as compared to 19% of controls.

There are common neural mechanisms involved in the different dependencies. Neuroscience tries to highlight the importance of functional and biochemical markers of susceptibility and indicators of changes in receptor response at the base of the dependencies themselves.

SIBioC NATIONAL SURVEY ON STAT TESTING IN CLINICAL LABORATORIES

G. Lippi

Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy

The term “stat” derives from the Latin word “statim”, and denotes a panel of diagnostic tests that are produced with a very short turnaround time, especially for patients in critical conditions (1). According to this unambiguous definition, stat tests should only be requested when necessary, in order to prevent abuse and overuse of laboratory resources. Another good reason for limiting stat testing is that urgent analyses are more expensive and seldom require dedicated personnel and instrumentation (2, 3).

Since scarce information is available on the burden of stat testing in clinical laboratories, especially in our Country, a specific web-based questionnaire was designed and disseminated to all the member of SIBioC, in order to obtain reliable information about the percentage of urgent analyses according to geographical location, dimension of the laboratory and healthcare setting.

After the deadline, a total number of 142 valid responses were collected (response rate ∼3.0%). The median percentage of urgent analyses over the number of total testing or the number of total inpatient testing were respectively 20% (IQR, 10-30%) and 33% (IQR, 11-50%). In the greatest part of Italy (i.e., more than 80%), the stat tests were comprised between 0 and 40% of the overall volume of testing, whereas less than 2% of laboratories performed 80% or more of the total testing volume as urgent. Interestingly, the percentage of urgent analyses was significantly higher in University facilities and much lower in private labs. Moreover, facilities with a testing volume <1 million of tests per year performed a percentage of stat tests consistently lower. The burden of stat analyses was also significantly higher in facilities located in the South and islands compared to north and center.

Despite a broad heterogeneity across the nation, the interesting data captured from this survey attests that the burden of stat testing in our Country is globally lower that that reported in other realities such as the US (i.e., 20% vs 37%) (4). It is noteworthy, however, that approximately 25% of all Italian laboratories currently perform more than half of the total testing volume as urgent analyses. Another important finding is that the overall prevalence of stat testing seems higher in the South and Islands (30%), intermediate in the North (20%) and lower in the Center (18%).

References

1.Lippi G, Caputo M, Banfi G, Buttarello M, Ceriotti F, Daves M et al. Recommendations for the detection and management of critical values in clinical laboratories. Biochim Clin 2008;32:209-16.

2.Scherrenburg J, van de Wijngaart DJ, Janssens PM. Reducing the number of clinical stat phlebotomy orders: feasible or not? Clin Chem Lab Med 2012;50:2141-8.

3.Lippi G, Simundic AM, Plebani M. Phlebotomy, stat testing and laboratory organization: an intriguing relationship. Clin Chem Lab Med 2012;50:2065-8.

4.Volmar KE, Wilkinson DS, Wagar EA, Lehman CM. Utilization of stat test priority in the clinical laboratory: a College of American Pathologists q-probes study of 52 institutions. Arch Pathol Lab Med 2013;137:220-7.

WHAT EMERGENCY DEPARTMENT WANTS FROM LAB.

G. Cervellin

Parma

The overcrowding in Emergency Departments (EDs) is a well known phenomenon in western countries, and it carries several potentially negative consequences. Overcrowding, amongst other factors, can be influenced by different timings of various care-paths (including the Turn-Around Time – TAT – of lab tests). It can influence the quality of care given to patients, and has also been considered as a first-line cause of doctor’s and nurse’s burn-out and of verbal or physical assault. The ED is a challenging environment, one of the most complex in healthcare, where clinical decision(s) must be rapidly undertaken to rule in or rule out a given disease, so assuring the appropriate care to the patients, and prevent overcrowding. It is hence of utmost importance the choice of the tests and appropriateness of the requests by Emergency Physicians (EPs), as well as the timeliness and the accuracy of the lab.

Although the ED is at greater risk of malpractice claims than several other healthcare setting, defensive medicine and redundancy of testing, are not effective choices, due to the negative consequences in terms of global diagnostic efficiency and resources utilization. In fact, in several clinical contexts, excessive testing can definitely be harmful for patients. Some practical decisions should be undertaken after weighting the implications of time pressure and ED overcrowding, and need to perform timely and accurate diagnosis or treatments. The simplest thing is often the best: observe your patient! When a given test is not sufficiently accurate to change the clinical management in a particular setting, it should not be prescribed. When asking a test the EP should consider that any molecule exhibits its own kinetic characteristics, which include the time of increase in blood or other fluids after disease onset, the relative increase over baseline, the peak concentration, the half-life and the time to return to normal. As such, the timing in requesting tests could be of crucial importance.

Since the concept that clinical judgement, based both on Bayesian reasoning and Gestalt perception, and laboratory testing should always go hand in hand, the topic of this presentation will be the discussion of diagnostic testing in the ED, as well as the proposal of some effective and efficient “prophiles”, tailored to some main presentations to the ED, such as chest pain, abdominal pain, major trauma, severe burns, dyspnea, stroke, hemorrhage, fever, arrhythmias, shock.

References

1. Carraro P. Esami di laboratorio raccomandati in alcune tipiche situazioni di Pronto Soccorso. Biochimica Clinica 2011;35:207-228.

2. Lippi G. Cervellin G, Plebani M. The ten commandments of laboratory testing for emergency physicians. Clin Chem Lab Med 2014;52:183-187.

3. Cervellin G, Borghi L, Lippi G. Do clinicians decide relying primarily on Bayesians principles or on Gestalt perception? Some pearls and pitfalls of Gestalt perception in medicine. Intern Emerg Med 2014; DOI 10.1007/s11739-014-1049-8 [Epub ahead of print].

4. Studdert DM, Mello MM, Sage WM, DesRoches CM, Peugh J, Zapert K, et al. Defensive medicine among high-risk specialist physicians in a volatile malpractice environment. J Am Med Assoc 2005;293:260917

5. Redberg R, Katz M, Grady D. Diagnostic tests: another frontier for less is more. Or why talking to your patient is a safe and effective method of reassurance. Arch Intern Med 2011;171:619.

THE REPLY OF LABORATORY

P. Carraro

Padova

The modern setting of health services is based on the centrality of the patient. In this direction, the organization of the clinical laboratory must respond in a timely manner and with appropriate test panels to the needs of the emergency room. The clinical side needs operational simplifications and so the test panels specific for the symptoms are a valuable contribution to a better care. The main frameworks in emergency presentation were examined by a document of recommendations specifically edited by SIBioC (1). Some clinical situations are here treated according to decision algorithms. By way of example, defining as an organic condition an acute dyspnea, the most important test is the arterial blood gas analysis (that is better than the pulse oximetry) and subsequently we can choose the appropriate tests on the basis of the single case characteristics. Based on symptoms and signs we can exclude a vascular dyspnea (after a pre-test score for pulmonary embolism), detect an inflammation status (leukocytes, CRP) or sepsis (procalcitonin and cultures), assess the peripheral perfusion (lactic acid), the metabolic state (electrolytes, urea, creatinine, glucose, bicarbonate and ketones), the different causes of heart failure (with Mg, K, natriuretic peptides, cardiac troponin), causes mechanical or traumatic (pCO2, hypoventilation), the loss of lung parenchyma in chronic exacerbated forms, thyrotoxicosis (TSH), dehydration (urea, sodium, potassium, urine and plasma osmolality) and other causes. This example demonstrates that the choice of laboratory test is complex and must be managed in a rational manner. A second example is the laboratory approach to syncope. The “ROSE study” (2) has recently proposed the acronym BRACES to better remember the tests to be performed in these cases. 3 of 6 examinations are laboratory tests, including the natriuretic peptides, hemoglobin and O2 saturation. These choices show a pragmatic approach to the definition of appropriateness, that depends on the clinical effectiveness rather on pathophysiological reasoning. This case demonstrates the changing of emergency medicine towards evidence-based medicine.

Finally, also other clinical conditions such as abdominal pain, the multiple trauma, disorders of consciousness are examined.

Reference

1. Carraro P, Study Group SIBioC: The laboratory for urgency/emergencies. Laboratory tests recommended in some typical conditions of the ER. Biochimica clinica 2011; 35:207-28.

2. Reed MJ, Newby DE, Coull AJ, et al. The ROSE (risk stratification of syncope in the emergency department) study. J Am Coll Cardiol 2010;55:713-21.

REASONS FOR INTEGRATING STAT AND ROUTINE LABORATORY TESTING

D. Giavarina

Clinical Chemistry and Hematology Laboratory, St. Bortolo Hospital, Vicenza, Italy

The main house and almost the only goal of STAT-laboratories is to provide a preferential way to the urgent samples, for a quicker response. STAT means test results transmission or availability within a defined and agreed time, according to clinical necessity. Turnaround time (TAT) is one of the most noticeable signs of laboratory service and is often used as a key performance indicator of laboratory performance. However, there are some differences in defining TAT. Laboratories most commonly define TAT as the time of specimen receipt in the laboratory to the time to reporting of results. Clinicians may define TAT from test order to reporting and many other definitions are available (1, 2).

The main causes of laboratory TAT delay can be ascribed to order entry, responsible for about 30% of all reports that are delayed. Sample collection causes 27% of delays, analytical phases 28%, report transmission 2%; the remaining 13% was not determined (3).

Most hospitals in the western world are “acute hospitals”, with a wide range of specialists and treatments for patients. Inside these hospitals, there are many outpatient services for chronic pathology or general medicine and surgery (oncology, nephrology, haematology, day surgery).

For both the “acute care” and outpatient offices, laboratory services must ensure a short TAT (usually less than 1 hour) to guaranteed timely patient care. In addition, outpatients being cared for by general practitioners require rapid response for test requests since most of these patients are affected by chronic conditions which require continuous monitoring in order to guide effective and prompt adjustments to therapy (i.e., Oral Anticoagulant Therapy, Diabetes, Hearth failure). Therefore, in the current healthcare system, most laboratory tests should be considered “STAT”.

Accordingly to these organizational models, there are essentially three ways to approach Stat tests: central laboratory testing, dedicated satellite laboratories and by POCT, using different workflow process with subsequent differences in timeliness.

We are now moving towards a new model of healthcare systems, where every patient has a critical pathology and treatment is “urgent” for everyone. In this scenario, laboratories need to consider every test as a Stat test, so that the separation between routine and stat organizations will be abolished. Automation, and particularly TLA represents formidable tools to meet the increasingly more demanding critical needs and, even more importantly, improve patient outcomes. POCT could be variously integrated into Stat test activities, usually in the evaluation of vital functions.

References

1. Breil B, Fritz F, Thiemann V, Dugas M. Mapping Turnaround Times (TAT) to a Generic Timeline: A Systematic Review of TAT Definitions in Clinical Domains. BMC Medical Informatics and Decision Making 2011, 11:34.

2. Pati HP, Singh G. Turnaround Time (TAT): Difference in Concept for Laboratory and Clinician. Indian J Hematol Blood Transfus 2014;30:81-84.

3. Steindel SJ, Novis DA. Using outlier events to monitor test turnaround time. Arch Pathol Lab Med 1999;123:607-14.

REASONS BEHIND THE AUTONOMY OF STAT LABORATORY

G. Pellegrini

Chemical-Clinical Analysis Laboratory, AOUP, Pisa, Italy

On the 30th of December 2010, in Pisa, when the Emergency Department was moved from Santa Chiara Hospital to Cisanello Hospital, a new urgent needs laboratory has been activated.

The activation of a new service is a very complex process: an effective and efficient planning has to meet the requirements of all the stakeholders (patients, hospital, suppliers) and all the singles phases have to be planned and monitored, through mandatory steps of review, verification and validation of the project.

In an initial stage has been carried out an analysis with the aim of separating the urgent exams-that require a short T.A.T (Turn-Around Time) and have to be transferred to the STAT /new laboratory-from the organisational urgencies, included in the clinical priority pathways, that have to be kept in the Routine Laboratory.

Three years after the opening of the laboratory, a series of surveys designed to assess the following aspects were carried out:

1. The intra-laboratory TAT, expressed as “outliers” (for TAT <60 ’ ), as the median to the 95th percentile of “key” analytes (Potassium, Prothrombin Time, Hemoglobin, Troponin T,);

2. Reduction of urgent test requests after the application of the ‘Pertinence Criteria’ according to D.L. 1235 of 30/12/2012 Region of Tuscany (IT block for inappropriate tests, guidelines and recommendations application, working groups etc.);

3. Evaluation of the level of satisfaction of TAT laboratory by Clinicians;

4. Comparative study about the efficiency of the STAT laboratory compared to the Routine Laboratory (expressed as the average cost test).

In conclusion, it can be stated that a STAT Laboratory to justify its existence has to ensure an appropriate mix of the two primary qualities of effectiveness and efficiency and has to be considered acceptable not just from the Medical Staff laboratory (interested in analytical quality, focusing on imprecision and inaccuracy goals), but also have to satisfy the needs and expectations of the Clinicians (interested in service quality, availability, cost and timeliness relevance).

References

1. Maria Salinas, Maite Lopez-Garrigòs, Mercedes Gutiérrez, Javier Lugo, Francisco Liorca and Joaquin Uris-Stat laboratory timeliness management according to clinician need. Clin. Chem. Lab. Med. 2011; 49 (2): 331-333

2. Giuliano Soffiati e Davide Giavarina-Stat laboratory testing: integration or autonomy? Clin. Chem. Lab. Med. 2010; 48 (7): 927-930

3. Adam J. Singer, Peter Viccellio, Henry C. Thode Jr., Jay L. Bock, Mark C. Henry-Introduction of a stat laboratory reduces emergency department length of stay. Acad. Emerg. Med. 2008; 15 (4): 324–328

4. Robert C. Hawkins-Laboratory turnaround time. Clin. Biochem. Rev. 2007; 28 (4): 179-194

5. Steven J. Steindel, Peter J. Howanitz-Physician satisfaction and emergency department laboratory test turnaround Time. Arch. Pathol. Lab. Med. 2001; 125: 863-871

THE IMPACT OF TOTAL LABORATORY AUTOMATION ON CORELAB EFFICIENCY SEEN THROUGH ITS EFFICIENCY IN MANAGING STAT REQUESTS: A 47 MONTHS EXPERIENCE AT THE UNIVERSITY HOSPITAL OF “TOR VERGATA”

C. Ialongo, I. Giambini, S. Bernardini

Department of Laboratory Medicine, University Hospital “Tor Vergata”, Rome

Background: Since its introduction in the mid-‘60s, the automation has been perceived like a true “industrial revolution” to the world of clinical laboratory. The growing success has led the automation to be pushed to an even higher level, with pre- (sample check-in, sorting, separation and delivery) and post-analytics (storage and disposition) assimilated in a functional continuum together with the analytical phase “sensu stricto”, and to which the name of Total Laboratory Automation (TLA) has been given.

Aim: Aim of this study is to evaluate the impact of TLA implementation on core laboratory efficiency, in terms of both efficiency (as turnaround time, TAT) and productivity (a percentage of acceptable tests at 45 minutes, PAT45) in the management of STAT requests

Materials and methods: a total of 143,539 STAT cardiotroponin I (CTNI) requests ordered between june 2010 and april 2014 were retrieved from the resident laboratory information system (LIS), of which 46,571 records (32.44%) considered as valid for this study.

The entity of TLA impact was analysed by a quasiexperimental design based on interrupted time series (ITS) and piecewise linear regression (PLR)

Results: TLA implementation produced a gradual and permanent reduction in the median TAT of STAT request, which was of 13.44 minutes for Emergency Department (ED) and of 25.74 minutes for non-Emergency Departments (NED). A considerable reduction was also noticed in the TAT of requests in overtime, with mean overtime TAT of 64.76 minutes for ED and of 76.25 for NEDs (respect to a 97.62 minutes and 122.90 minutes before TLA respectively). ED’s PAT45 also increased of 31.07 percentage points, reaching the 63.35% of completed requests within 45 minutes after TLA implementation. Noticeably, effects of TLA became fully evident in about one 1 year for ED requests, and in just 4 months for NEDs.

Conclusions: TLA can be considered able to manage the STAT requests in a fully-integrated workflow with the core laboratory routine activity. Notably, it can satisfy the needs of a large university hospital, for both outpatients and inpatients urgent requests. Finally, TLA effects become evident after a certain time after the implementation, in dependence of the former level of workflow control, and remain almost stable in the long term.

FILMARRAY SYSTEM VERSUS RT-PCR METHOD IN MENINGITIDIS AND SEPSIS MANAGEMENT: AN EXAMPLE OF ROUTINE-EMERGENCY INTEGRATION

L. Bianchi, Z. Napoli, S. Donati, S. Bovani, P. Lencioni, R. Lari

Lab. Analisi Chimico-Cliniche e Microbiologiche, Osp. S. Jacopo, Pistoia

Introduction: Meningitis and bacterial sepsis requires a multidisciplinary approach and an emergency/routine regimen (1).

Objectives: Aim of this study was to evaluate: 1) Sensitivity, Negative Predictive Value (NPV) and Positive Predictive Value (PPV) of Real-Time PCR (RT-PCR; Eurospital, DID) and FilmArray technology (TFA; Biofire, DID) on cerebrospinal fluid (CSF) and blood sample (BS); 2) efficacy of RT-PCR and TFA in sepsis and meningitis management by evaluation of Turn Around Time (TAT) compared to classic methods.

Methods: 240 CSF, 180 BS and 9 cavitary liquids (CL) were tested with RT-PCR (Biomerieux; Eurospital) and traditional bacteriological methods like Gram stained bacterioscopic examination (GS), cultural examination (CE) and soluble antigen extraction (AE) for CSF. RTPCR positive samples (29 CSF, 25 BS and 5 CL)were tested with TFA.

Results: All negative RT-PCR CSF resulted negative to traditional methods (GS, CE and AE). 12 positive RT-PCR CSF resulted TFA negative; these samples showed a Cycle Threshold (Ct) >30 with negative CE in 75% of cases. On BS the sensitivity values of blood culture (BC), RT-PCR and TFA were 94,2%, 40,4% and 44,2% respectively; considering only detectable pathogens, sensitivity was 94,2%, 91,3% and 46%. TFA and RT-PCR, compared with BC, showed a PPV of 100%.

TFA and RT-PCR NPV were 80% and 79% respectively. CE in all matrices showed a TAT of 48-96h. RT-PCR and TFA showed for CSF and BS a TAT of 2-3h and 1h respectively. TFA, in case of sepsis, reduced TAT of 4-12h compared to BC.

Conclusions: RT-PCR is the most sensitive and specific method for sepsis and meningitis management and it allows to detect the causative agent directly on blood with a reduction in TAT compared to BC. RT-PCR shows a PPV on CSF and BS equal to 100%. Considering only detectable pathogens in BS, RT-PCR, compared to BC, shows a reduction in terms of sensitivity (92% VS 79%). On CSF TFA shows the highest sensitivity compared to bacteriological methods. TFA, in CSF management of meningitis emergency, results an effective technique in terms of test preparation (2 min), TAT (1h) and result interpretation. TFA and RT-PCR will be implemented for sepsis management in BS and in CL.

Reference

1. Brouwer et al. J Clin Microbiol 2010;42:2919-25.

CURRENT STATE OF DIAGNOSTIC TECHNOLOGIES FOR AUTOANTIBODY MEASUREMENT: UNDER THE SIGN OF AUTOMATION

R. Tozzoli

Department of Laboratory Medicine, S. Maria degli Angeli Hospital, Pordenone, Italy

Analytical systems for measuring autoantibodies (aAbs) have evolved in the last 20 years, encompassing 3 generations of immunoassay (IMA) methods. Two main categories of methods (morphologic and immunologic) have found applications in the diagnosis of autoimmune diseases (AIDs): starting from indirect immunofluorescence (IIF), a wide number of different IMAs were used for the single or multiple measurement of aAbs (monoplex and multiplex IMAs). The large increase in test requests occurred in the same years was accompanied by the introduction of automation in the clinical immunology laboratory: according to the evolution of diagnostic technologies, several of these methods have been the subject of total automation of the analytical procedure (1).

The automation of IIF. Following the statement that the IIF method should be considered as the standard method for the detection of aAbs, in particular for the so-called anti-nuclear antibodies (ANA), because of its sensitivity enabling recognition of >100 autoantibody targets), the recent evolution of commercial systems is based on the automation of slides preparation and microscope reading (2). The innovation is based on the digitalization of images and on the classification of patients using computer-aided approaches: literature studies showed their high efficiency in discriminating between positive and negative samples. Currently, at least 6 commercial automated systems are available: Aklides (Medipan); Europattern (Euroimmun); Helios (Aesku Diagnostics); Nova View (Inova Diagnostics); Image Navigator (ImmunoConcepts), Zenit G-sight (Menarini Diagnostics).

The advent of automated monoplex platforms. Starting from 1995 automated monoplex IMA platforms have progressively substituted the manual radio- and enzyme-immunoassays. A wide range of labels and label detection systems are developed, i.e. fluorophores (fluorescent IMA) and luminophores (chemiluminescent IMA-CLIA). Currently, at least 9 automated CLIA monoplex platforms are available for the quantitative measurement of autoantibodies. The newer systems are BioFlash (Inova Diagnostics, California) and Zenit RA (Menarini, Italy), and particularly Maglumi (SNIBE, China). Several literature reports show the high correlation of the results of these platforms with those obtained with manual IMA methods.

The progressive diffusion of the multiplex methods. The advent of multiplex proteomic technologies seems to be an optimal solution for the parallel measurement of different aAbs: some of these microarrays (planar and non-planar IMA) may contribute to evercome some drawbacks of the monoplex IMA (time, costs, lack of harmonization, volume of reagents/samples, turn-around time, etc) (1), allowing the autoantibody profiling of AIDs patients for diagnostic and prognostic purposes. Between the different available solutions, the microsphere technology, in combination with flow cytometry, has been widely used for different aAbs in different AIDs. In the last 8 years, a multiplex fully-automated platform (BioPlex 2200, BioRad Laboratories, California) has been introduced in several laboratories in the world, originating a wide number of publications that highlighted its high diagnostic accuracy (3).

References

1. Tozzoli R, Bonaguri C, Melegari A, et al. Current state of diagnostic technologies in the autoimmunology laboratory. Clin Chem Lab Med 2013;51:129-38.

2. Bizzaro N, Antico A, Platzgummer S, et al. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems. Autoimmun Rev 2014;13:292-8.

3. Tozzoli R, Villalta D. Autoantibody profiling of patients with antiphospholipid syndrome using an automated multiplexed immunoassay system. Autoimmun Rev 2014;13:59-63.

AUTOIMMUNITÀ E VITAMINA D: QUALI EVIDENZE?

L. Andreoli

Brescia

There has been increasing interest for the relationship between vitamin D (vit.D) and the modulation of the immune system, with particular reference to autoimmune diseases.

Epidemiological data underline a strong correlation between poor vit.D status and higher risk for chronic inflammatory illnesses of various etiologies, including autoimmune diseases. Epidemiological, genetic, and basic studies indicated a potential role of vit.D in the pathogenesis of certain systemic and organ-specific autoimmune diseases. These studies demonstrate correlation between low vit.D and prevalence of diseases (1).

Serum levels of vit.D have been found to be significantly lower in patients with systemic lupus erythematosus, undifferentiated connective tissue disease, and type-1 diabetes mellitus (DM1) than in the healthy population. In addition, it was also found that lower levels of vit.D were associated with higher disease activity in rheumatoid arthritis (2).

1,25-Dihydroxyvitamin D [1,25OH-vit.D], the biologically active form of vitamin D regulates the growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory properties. Cells involved in innate and adaptive immune responses--including macrophages, dendritic cells (DCs), T cells and B cells--express the vit.D receptor (VDR), and can both produce and respond to 1,25OH-vit.D (3).

A cell population of particular interest are DCs, because they induce or tolerize T cells, and tolerogenic DCs can promote the development of regulatory T cells (Treg) with suppressive activity. DCs are key targets for the immunomodulatory effects of VDR agonists that enhance their tolerogenicity in adaptive immune responses. Tolerogenic DCs induced by a short treatment with VDR agonists promote CD4+CD25+Foxp3+ Treg cells that are able to mediate transplantation tolerance and to arrest the development of autoimmune diseases. Thus, the possibility of manipulating DCs with VDR agonists could be exploited to control a variety of chronic immuno-mediated inflammatory conditions (4).

Therefore, there is great interest in clinical translation of the potential multiple benefits of vit.D. Most of the human studies so far have applied supplementation with the pre-active vit.D (25-OH vitamin D), a molecule that has a very good safety profile even at high doses. A recent systematic review of the literature reported that cross-sectional studies confirm that levels of vit.D <30 ng/mL are present in a significant percentage of patients with autoimmune disease (30- 77%), and link profound deficiency (<10 ng/mL) with aggravation of symptomatology, while genetic studies associate polymorphism of VDR to various autoimmune diseases. Among experimental studies on humans, only those on DM1 prove that the risks are significantly reduced in infants treated with vit.D after the 7th month (OR 0.71, 95% CI, 0.60 to 0.84) and that a dose-response effect exists (5).

Overall, there is enough evidence to support the potential benefit of vit.D supplementation in patients with chronic autoimmune diseases. The impact of this safe, cheap and well-tolerated drug intervention should be established in randomized clinical trials.

References

1. Agmon-Levin N, Theodor E, Segal RM, Shoenfeld Y. Vitamin D in systemic and organ-specific autoimmune diseases. Clin Rev Allergy Immunol. 2013;45(2):256-66.

2. Cutolo M, Plebani M, Shoenfeld Y, Adorini L, Tincani A. Vitamin D endocrine system and the immune response in rheumatic diseases. Vitam Horm. 2011;86:327-51.

3. Adorini L, Penna G. Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol. 2008;4(8):404-12.

4. Adorini L, Penna G. Dendritic cell tolerogenicity: a key mechanism in immunomodulation by vitamin D receptor agonists. Hum Immunol. 2009 May;70(5):345-52.

5. Antico A, Tampoia M, Tozzoli R, Bizzaro N. Can supplementation with vitamin D reduce the risk or modify the course of autoimmune diseases? A systematic review of the literature. Autoimmun Rev. 2012;12(2):127-36.

LABORATORIO E DIAGNOSIS DI CELIACHIA: UPDATE 2014

A. Fasano

Boston

Celiac disease (CD) is a systemic immune-mediated condition triggered by gluten in genetically susceptible individuals and affects about one percent of population worldwide (1-4 Snyder). Although in recent years great progress has been made in the diagnosis and management of CD (1), important problems still exist, partially due to the changes in its clinical presentation. More patients are diagnosed with less severe symptoms, and up to 40% are clinically silent (1). Anemia is a common finding in 8% of patients, while there is a decrease in the number of patients presenting with the classic symptom of diarrhea (43% now compared with 73% before 1993) (2). In fact, many patients may be misdiagnosed, with 5-30% of patients having been at some point diagnosed with irritable bowel syndrome (3). Therefore, accurate diagnostic screening and confirmatory tests are of paramount importance. Several tests are utilized to diagnose and monitor CD, including serologic tests, genetic testing and histology (1). The current diagnostic algorithm for CD includes initial screening serological tests, followed by a confirmatory small intestinal biopsy showing the autoimmune insult typical of CD in children and adults (1). The relative merits of these tests in various situations are discussed below.

Serology. Measurement of IgA antibodies to tissue transglutaminase (anti-tTG) is recommended for initial testing, due to high sensitivity (94%) specificity (97%), and excellent standardization (1). The IgA anti-endomysium (EMA) determination is nearly 100% specific of active CD, but should be used only as a confirmatory test and in special cases in which anti-tTG can be falsely positive (other autoimmune diseases like type 1 diabetes and autoimmune hepatitis) since it is expensive and operator-dependent (1). More recently, deamidated gliadin peptides (DGP) antibodies, particularly of the IgG class, have been introduced with a sensitivity and specificity comparable to IgA anti-tTG, but with a better performance in IgA-deficient subjects and in children younger than three years (4). A recent study summarized the evidence from 2004 to September 2009 on the performance of laboratory-based serological tests for diagnosing CD in children, using histology as the reference standard (5). A total of 16 articles were included in a meta-analysis, reporting on 3110 patients (1876 with CD, 1234 without CD). The results showed that IgA-EmA and IgA-anti-tTG tests appear highly accurate to diagnose CD. IgG-anti-DGP tests may help in excluding CD. IgA-anti-gliadin antibody (AGA) and IgA-DGP tests showed inferior accuracy (5).

Histology. A small intestinal biopsy is required in most patients with suspected CD for final diagnosis. The characteristic histological changes include an increased number of IELs (>25 per 100 enterocytes), elongation of the crypts, and partial to total villous atrophy (1). However, the biopsy, long considered the diagnostic gold standard, has been recently questioned as a reliable and conclusive test for every case (1). Indeed, the wide variability of CD- related findings suggests that it is difficult to conceptualize the diagnostic process into rigid algorithms that do not always cover the clinical complexity of this disease (1).

References

1. Fasano A, Catassi C. Clinical Practice: Celiac disease. N Engl J Med 2012;367:2419-26.

2. Lee SK, Green PH. Celiac sprue (the great modern-day imposter). Curr Opin Rheumatol 2006;18:101-7.

3. Sanders DS, Carter MJ, Hurlstone DP, Pearce A, Ward AM, McAlindon ME, Lobo AJ. Association of adult coeliac disease with irritable bowel syndrome: A case-control study in patients fulfilling ROME II criteria referred to secondary care. Lancet 2001;358:1504-8.

4. Rostam A, Murray JA, Kagnoff MF. AGA technical review on the diagnosis and management of celiac disease. Gastroenterology 2006;131:1981-2002.

5. National Institutes of Health Consensus Development Conference. Statement on celiac disease, June 28-30, 2004. Gastroenterology 2005;128(4 Suppl 1):S1-9.

DIAGNOSTIC EFFICIENCY AND EFFICACY AND LABORATOY COMPETENCE: ANA REFLEX TEST

C. Bonaguri¹, A. Melegari²

¹Diagnostic Laboratory Department, Parma Hospital, Parma, Italy

²Diagnostic Laboratory Department, NOCSAE Hospital, Modena, Italy

Introduction: The limitation of inappropriate test requests and the identification of a balance between available economic resources and increasing health needs is crucial for modern Healthcare Services. In this perspective, harmonization of testing algorithms is a goal. We have specifically focused on antinuclear antibodies (ANA), Extractable Nuclear Antigens (ENA) and double-stand (dsDNA) (1). A previous study performed in North Western area of Emilia Romagna district (AVEN) showed that development and implementation of an algorithm for the diagnostics of Autoimmune Rheumatic Disease in hospitalised patients was associated with both a reduction in number of second-level tests and an increased diagnostic specificity (2). The Emilia-Romagna Region, on March 1^th, 2013 has released the first coding for ANA Reflex testing with the aim to promote an appropriate use of laboratory resources. ANA Reflex is a common guideline for autoantibody testing in Autoimmune Rheumatic Disease, which places ANA test at the first analytical level whereas the following steps are directly guided by the laboratory. This diagnostic algorithm was been implemented in all Autoimmune Laboratories of Emilia-Romagna Region.

Methods: We have compared both the number of ANA, ENA and dsDNA tests and the percentage of positive results before and after the implementation of ANA Reflex (May 2012-April 2013 vs May 2013-April 2014) in Autoimmune Laboratories of Parma and Modena.

Results: During the first year of ANA Reflex implementation we have observed that the requests of ANA Reflex represented a significant percentage of total ANA requests in both Parma and Modena (46% and 49% respectively). We have also found that ENA requests during this period showed a reduction of 22% in Parma and 21% in Modena, accompanied by a substantial increase of positivity. DNA showed a reduction of 14% in Parma and 26% in Modena before and after implementation of ANA Reflex, with a substantial improvement of selected positive cases.

Conclusions: Appropriateness is an important objective for Healthcare Services. The clinical laboratories may provide useful diagnostic tools in order to improve the clinical decision-making in the field of Autoimmune Diseases. In our experience, the strict cooperation between clinicians, laboratory specialists and Healthcare Services was crucial to the diffusion of ANA Reflex testing. Moreover, efficiency and efficacy were strongly linked: the percentage of second level test positivity has increased both for ENA and dsDNA after implementation of ANA Reflex test.

References

1. Wiik A.S. Diagnostic strategies for autoimmune rheumatic diseases. Z.Rheumatol. 2007;66(3):219-220.

2. Bonaguri C., Melegari A., Ballabio A. et al. Italian multicentre study of a diagnostic algorithm in autoantibody testing for autoimmuni rheumatic: Conclusive results. Autoimmun.Rev. 2011;11:1-5.5.

THE NEW HELIOS PLATFORM: EVALUATION OF ANTINUCLEAR ANTIBODIES (ANA) AND ANTI NEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA)

M. Seguso, N. Gallo, M. Arzenton, S. Dainese, M.G. Epifani, M. Plebani

U.O.C. Medicina di Laboratorio, Az. Osp.-Università degli Studi, Padova

Aim: To compare the new Helios platform (Aesku Diagnostics, Wendelsheim, Germany) with the well established indirect immunofluorescence (IIF) method, in discriminating Antinuclear Antibodies (ANA) and Anti neutrophil cytoplasmic antibodies (ANCA).

Methods: We examined 724 unselected sera from our routine for ANA. The samples were determined with the IIF method on Hep-2 cells (Inova Diagnostics, USA) and with Helios system (Hep-2 cells Aesku). Employing IIF, ANCA were detected by running 141 patient samples on ethanol-fixed neutrophils and formalin-fixed neutrophil according to the recommendations of the manufacturer (Inova Diagnostics, USA) and with Helios system. Every sample was confirmed with fluorenzymeimmunoassay (FEIA) method. IIF results were evaluated independently by two operators and by Helios reader.

The automatic reader Helios analyzed four (for ANA) or three images (for ANCA) per well and assigned the positivity when at least three (for ANA) or two images (for ANCA) were positive.

Results: -ANA:The operators classified 382 samples as negative, 168 samples as low titres (1:80) and 190 as positive. Helios System correctly classified 372/382 negative samples and 143/174 positive samples with 86% sensitivity and 96% specificity. Discrepant results regarded mainly samples falling in the low titre category and this might depend on: 1.differences in substrate type used; 2.differences in cut-off setting. Moreover, it should be emphasized that in our laboratory routine workflow, low titre samples are frequently encounter and they have been rarely taken into account in previous validation studies. (Bizzaro N. et al, Autoimmun Rev 2014)

-ANCA:The operators classified 93 samples as positive and 48 samples as negative. Helios System correctly recognized 38/48 negative samples and 85/93 positive samples with 89.5% sensivity and 82.6% specificity. If we considered the measurement of PR3 and MPO (FEIA method) Helios showed a good agreement.

Conclusion: We found that x-ANCA could be easily identify with Helios platform but we still need more studies. Automated interpretation systems such as the Helios platform may reduce intra and inter-laboratory variability, improve laboratory efficiency and support standardization of IIF especially in high throughput laboratories.

DISCRIMINATION OF PSEUDODEMENTIA FROM ALZHEIMER’S DISEASE USING CSF BIOMARKERS

G.M. Sancesario¹, C. Liguori², M. Nuccetelli¹, A. Martorana^2,3, G. Sancesario^2,3, S. Bernardini¹

¹Dept. of Experimental Medicine and Surgery, University of Rome “Tor Vergata”

²Dept. Neuroscience, University of Rome Tor Vergata

³IRCCS Santa Lucia Foundation, Rome

Depression and dementia, often in the form of Alzheimer’s disease (AD), can occur together in the elderly and may be confused each other (1,2). In the depressed elderly, depression itself can affect cognitive performances, diagnosed as pseudodementia secondary to depression, which is an independent process from cognitive impairment caused by dementia (3). However, depression could be an early symptom or a risk factor of AD, complicating the early and correct diagnosis (4). Biomarkers can help to identify possibly treatable patients to reverse underlying causes of mood disorder.

We evaluate the level of baseline CSF biomarkers β-amyloid₄₂ (Aβ42), total Tau (T-Tau) and Phospo-tau181 (P-Tau) in individuals that were admitted to the Neurological Centre of the Tor Vergata General Hospital between 2009-2011 and retrospectively divided in three groups: patients suffering of pseudodementia (n=9), AD (n=12) and other neurological disorder (n=14) on the base of deterioration (AD) or stability/improvement (pseudodementia) of cognitive functions after two-three years follow-up. Interestingly, the level of Aβ42 in pseudodementia patients was significantly higher than in AD group (749.10±199.30 vs 322.50±87.51 pg/mL, respectively), and similar to control group (862.90±230.40 pg/mL); moreover, both T-Tau and P-Tau were lower in pseudodementia patients than in AD (185.10±59.49 vs 712.70±327.20 pg/mL for Total-Tau and 32.56±8.09 vs 76.83±24.01 pg/mL for P-Tau, respectively) and similar to control (231.10±78.42 pg/mL for T-Tau and 40.50±11.81 pg/mL for P-Tau).

These new findings demonstrate that the CSF biomarkers can clearly discriminate patients suffering of pseudodementia from AD patients. A timely diagnosis of dementia or pseudodementia is crucial to the choice of treatment strategy.

References

1. Ownby RL, Crocco E, Acevedo A, et al. Depression and risk for Alzheimer disease: systematic review, meta-analysis, and metaregression analysis. Arch Gen Psychiatry 2006;63:530-8.

2. Kobayashi T, Kato S. Depression-dementia medius: between depression and the manifestation of dementia symptoms. Psychogeriatrics 2011;11:177-82.

3. Wilson RS, Mendes de Leon CF, Bennet DA, et al. Depressive symptoms and cognitive decline in a community population of older persons. J Neurol Neurosurg Psychiatry 2004;75:126– 9.

4. Schweitzer I, Tuckwell V, O’Brien J, et al. Is late onset depression a prodrome to dementia? Int J Geriatr Psychiatry 2002;17:997-1005.

THE IMPORTANCE OF INDIVIDUAL BIOLOGY IN THE CLINICAL USE OF SERUM BIOMARKERS FOR OVARIAN CANCER

F. Braga, M. Panteghini

Scuola di Specializzazione in Biochimica Clinica e Cattedra di Biochimica Clinica e Biologia Molecolare Clinica, Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, University of Milan Medical School, Milano, Italy

Background: An increased focus on the biological behaviour of biomarkers for ovarian cancer (OC), i.e., carbohydrate antigen 125 (CA-125) and human epididymis protein 4 (HE4), has been advocated to improve their clinical use (1). Due to the paucity and poor design of available studies evaluating biological variation (BV) of CA-125 and the lack of BV data for HE4, in this study we evaluated BV of both biomarkers by adopting an accurately experimental protocol.

Methods: We monthly obtained serum samples from 28 apparently healthy women (14 pre- (PreM) and 14 post- menopausal (PostM), ages 25-68 years) for four consecutive months. Special attention was paid to pre-analytical sources of variability. Once all samples were available, they were analyzed in a single run in duplicate for CA-125 and HE4 on Roche Modular system by electrochemiluminescence immunoassays. Cochran’s test was performed for outlier identification among observations and within-subject variances, whereas Reed’s criterion was used for identification of outliers among mean values of subjects. A Shapiro-Wilk test was applied to the set of results from each individual and to values of all subjects, and PreM and PostM groups separately, to check normality of data distribution. Data were analyzed by the ANOVA (2).

Results: After outlier exclusion, the suitable subjects for BV estimate were 27 (13 PreM and 14 PostM) for CA-125 and 28 for HE4. The Shapiro-Wilk test accepted the hypothesis of normality for the distribution of the majority of intra- individual marker values (100% for CA-125 and 89% for HE4). The HE4 values in the whole group of women were normally distributed, while those of CA-125 failed the normality test. Therefore, while HE4 statistics were directly performed on raw data, for CA-125 a natural log-scale transformation was applied. Before deriving biological CVs the log-transformed variances and overall mean were converted back via inverse natural log function. For both biomarkers no difference in median concentrations was found between PreM and PostM. For CA-125 the intra-individual CV (CVI) was not different between groups (9.1% in both). For HE4 CVI was higher in PreM (12.1%) than in PostM (6.5%) (P<0.001). Between-subject CVs were 10.6% for CA-125 and 16.4% for HE4, with no influence by the fertility status. Both biomarkers showed high individuality meaning that the use of population-based reference limits may have limited value for their interpretation. Reference change values (RCV) were 26% for CA-125 (all), 34% for HE4 (PreM) and 18% for HE4 (PostM). Desirable analytical goals for imprecision, bias, and total error were <4.5%, ±3.5% and ±11.0% for CA-125, <6.0%, ±5.0% and ±14.9% for HE4 PreM and <3.2%, ±4.7% and ±10.0% for HE4 PostM.

Conclusions: Monitoring longitudinal changes in serum concentrations of OC biomarkers over time is probably better than using single-threshold rules. According to differences in BV due to the hormonal status, one should differently interpret HE4 changes in PreM and PostM. Conversely, the interpretation of CA-125 concentrations does not require different approaches.

References

1. Ferraro S, Panteghini M. Is serum human epididymis protein 4 ready for prime time? Ann Clin Biochem 2014;51:128-36.

2. Braga F, Ferraro S, Mozzi R, et al. Biological variation of neuroendocrine tumor markers chromogranin A and neuron-specific enolase. Clin Biochem 2013;46:148-51.

IMMUNOSUPPRESSIVE MYELOID CELLS IN PANCREATIC CANCER

E. Gnatta

Department of Medicine – DIMED, University of Padova, Italy

Established tumors are complex masses that contain not only neoplastic cells but also non-transformed cellular elements such as stromal cells and immune cells. In contrast to the case of a productive immune response to a pathogen, immune cells that reside within tumors are dysregulated and functionally impaired. An altered function of lymphocytes, dendritic cells (DC) and immature myeloid cells (IMCs) appears to be a hallmark of tumor-mediated immune suppression (1).

Immune cell alterations detected in pancreatic ductal adenocarcinoma (PDAC) patients may be the consequence of a direct or indirect crosstalk between cancer cells and the immune system, and this may occur within neoplastic lesions as well as at a systemic level. Soluble mediators directly secreted by tumor cells and/or indirectly produced by stromal inflammatory cells surely represent key players in this crosstalk. Moreover alterations in the expression of immunomodulatory molecules expressed on the surface of immune cells can be involved: cytotoxic T lymphocyte antigen 4 (CTLA4) is believed to participate in the immunosuppressive activity of regulatory T cells (Tregs) while programmed death ligand 1 (PDL1) is expressed by cancer cells as well as by cellular components of the tumor microenvironment and its expression by malignant cells appears to be of prognostic significance in PDAC patients. To get further insights into this issue, we studied the effects of culture media conditioned by PDAC cell lines exhibiting different metastatic potential on immune cells (2). Well-differentiated and poorly metastatic PDAC BxPC3 cells induced the expansion of CD33+CD14–HLA-DR– myeloid derived suppressive cells (MDSCs) while downregulating the expression of CTLA4 on their surface. Conversely, poorly differentiated and highly metastatic Capan1 cells not only promoted the expansion of CD33+CD14+HLA-DR– monocytic MDSCs, but also caused a reduction in CTLA4 and an increase in PDL1 expression levels in all IMCs considered.

A variety of cancer cell- and immune cell-derived soluble proteins may be responsible for the effects of PDAC conditioned media on immune cells. We focused on the S100A8/A9 complex because it has been involved in the establishment of a favorable environment for metastases, implicated in the recruitment of MDSCs and the stimulation of their immunosuppressive functions and shown to be intimately link with the deletion of SMAD4. When SMAD4 is deleted, PDAC cells acquire the ability to express high levels of the S100A8/A9 complex. S100A8/A9 caused an expansion of CD33+CD14+HLA-DR–monocytic MDSCs while reducing the levels of CTLA4 on their surface. As it failed to modulate CD33+CD14– HLA-DR– MDSCs, these findings suggest that the S100A8/A9 complex constitutes one of the immunosuppressive soluble mediators released by advanced PDACs. Thus, we suggest that the accumulation of genetic alterations by PDAC cells is paralleled by a progressive imbalance between regulatory and effector immune cells, ultimately resulting in the establishment of immunosuppressive networks that allow for rapid tumor progression.

References

1. Hanahan D, et al. Cell 2011.

2. Basso D, et al. PLoS One 2013.

DIAGNOSTIC IMPACT OF POLYCHROMATIC FLOW CYTOMETRY IN NON-HODGKIN LYMPHOMA

F. Visconte, M. Mattioli, R. Rosamilio, M. Raia, G. Scalia, L. Del Vecchio, L. Panico

Scuola di Specializzazione in Biochimica Clinica, Università Federico II, Napoli

Background: The diagnosis of non-Hodgkin lymphomas (NHL) is essentially based upon histology and immunohistochemistry (IHC). However, a series of so-called ancillary technologies are used to improve the accuracy of NHL identification and characterization.

We studied, by flow cytometry (FCM) on surgical node biopsies, a series of 228 cases of NHL belonging to various classes according to WHO classification. Our series included: small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/LLC, 13 cases), Nodal Marginal Zone Lymphoma (NMZL, 15), Follicular Lymphoma (FL, 39), Mantle Cell Lymphoma (MCL, 6), Diffuse Large B Cell Lymphoma (DLBCL, 46), Primary Mediastinal Large B Cell Lymphoma (PMLBCL, 2), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN, 1), Dendritic Cell Sarcoma (DCS, 1), Anaplastic Large Cell Lymphoma CD30+ (ALCL CD30+, 3), B-T-Cell Rich Lymphoma (B-T Rich, 2), Myeloid Tumor (MT, 2), Multiple Myeloma (MM, 1), Composite Lymphoma (CL, 1), T-Cell Lymphoma (TCL, 4), Metastasis (Met, 10), Reactive Hyperplasia (RH, 82).

Methods: We started from the concept that the most relevant features evidenced by FCM in NHL are: 1) asynchronous expression of T cell antigens and TCR-V restriction in T-cell NHL; 2) anomalous assembly of B cell antigens and κ/λ restriction in B-cell NHL; 3) absence of features at point 1 and 2 in T-cell and B-cell hyperplasia; 4) absence of CD45 expression in epithelial tumors. Independent classification of NHL is NOT an aim of FCM. The main goal of this study was to assess, on a non-competitive basis, the role of FCM in implementing accuracy of IHC diagnosis in NHL. The procedure is the following: 1) surgical biopsy is sent to Pathology Department; 2) the Pathologists gives his first look and, if appropriate, send a fragment to FCM lab (Hodgkin’s Lymphomas and Met are excluded); 3) FCM lab develops a complete T and B cell typing; 4) FCM lab sends the report to the Pathologist; 5) the Pathologists, combining his first look with FCM results gives his second look and proceeds to final typing (e.g. further IHC staining, detection of oncoproteins); 6) The Pathologist sends his final report to FCM lab and sends a combined (IHC/FCM) report to the Clinical Division.

Results: IHC was accurate in 219/228 cases (96%). FCM was accurate in 216/228 cases (94.7%). In the 9 cases in which IHC was inaccurate, the contribution of FCM was critical, allowing a combined, final diagnosis. In particular, these cases were 1 SLL, 3 NMZL, 1 CL, 1 TCL, 3 RH. Thus the final contribution of FCM to an accurate diagnosis was 3.9% (9/228). By contrast, cases in which FCM was unable to help IHC in producing an accurate diagnosis were 12 (5, DLCBL, 3 ALCL, 1 TCL, 2 B-T rich and 1 Met).

Conclusion: This study, for the first time, demonstrated that almost 4% of cases of NHL can be misdiagnosed by IHC alone, so that an ancillary technique, as FCM, is mandatory.

KRAS: ONE ACTOR, MANY POTENTIAL ROLES IN DIAGNOSIS

L. Bruno¹, F. Barbera¹, L. Barresi², C. Bellia³, B. Lo Sasso³, G. Bivona³, Alessia Pivetti³, P.G. Conaldi¹, M. Ciaccio³

¹Department of Laboratory Medicine and Advanced Biotechnologies-ISMETT-Palermo

²Gastroenterology and Endoscopy Unit-ISMETT-Palermo

³Department of Biopathology and Medical and Forensic Biotechnologies, University of Palermo

Background: Activating mutations in KRAS oncogene are often found in human tumors and are used as different diagnostic tools. Cyst fluid analysis is an effective method of distinguishing mucinous from non-mucinous cysts and to detect malignancy in pancreatic cystic lesions with significant implications in clinical intervention and follow-up.

Previous studies reported that KRAS mutations alone had a sensitivity of 45% and specificity of 96% for diagnosing mucinous cysts (Khalid A et al. 2009). The aim of this study was to compare the accuracy of molecular evaluation (KRAS status) and both tumor and biochemical markers, and to assess whether these diagnostic tools can be considered complementary to pancreatic cyst fluid classification.

Methods: Forty-seven patients with cyst of pancreas, undergoing at endoscopic ultrasound (EUS) with fine needle aspiration (FNA) at ISMETT (Palermo, Italy), were evaluated by cyst fluid analysis. Direct Automated Sequencing for exons 2-3 of KRAS gene, and both tumor and biochemical marker levels (CEA>192 ng/mL, CA19.9, lipase) were evaluated with usual diagnostic kits. A comparative analysis was performed between KRAS mutational status, endoscopic and cytologic evaluation, and both tumor and biochemical markers

Results: We performed cyst fluid DNA analysis in 47 patients with endoscopic and histopathologic diagnosis. KRAS mutations were detected in 18/47 patients (38%), and mutation in codon 61 was found in 3 patients with low CEA levels. All patients with KRAS mutation had a mucinous histology, and at least one of the tumor and/or biochemical parameters altered.

Conclusions: There is no single test to make a sure and accurate diagnosis in every pancreatic cystic lesion, but many diagnostic informations must be related to each other like pieces of a puzzle (Barresi L et al, 2012). Our data show that KRAS mutational status is a useful diagnostic tool for a better classification of pancreatic cysts with high malignant potential, but mutational analysis should also be extended to exon 3, to date not investigated in diagnostics. Therefore KRAS status can be considered a piece of this puzzle. Tumor markers, expecially CEA, are still useful in absence of KRAS mutations.

SALIVARY CORTISOL AND TESTOSTERONE CONCENTRATIONS DURING TRAINING AND REGULAR COMPETITIVE MATCH

G. Rapisarda¹, S. Ricca¹, S. Rizza², M. Lenzo¹, M. Scuto², A. Trovato Salinaro², V. Calabrese², R. Ientile¹

¹Department of Biomedical Sciences, University of Catania

²Department of Biomedical Sciences and Morpho-functional Imaging, University of Messina

Psychological and social stress are commonly associated with increases in cortisol levels (1). However, cortisol plays an important role, in physical exercise where it mobilizes energy stores and increased levels are associated with the intensity and duration of exercise as well as training levels of subjects (2). Variations in testosterone secretion have also been described during acute exercise and the performance in various sports.

Although there are conflicting results in the literature, it has been shown that for some athletic activities, both cortisol and testosterone concentrations might be related to physical performance, probably due to their influence on energy metabolism and/or behavior.

We aimed to examine salivary cortisol and testosterone concentrations in semi-professional female athletes of water polo, taking place within a competitive day, and to determine whether the concentrations of these hormones could be accurate predictors of performance for this sport.

Thirteen saliva samples were taken on a competition day as well as on a rest day according to the indicated times: one sample after awakening at 8:00 h, and then immediately after competition (16:00 h). The collected samples in the rest day was based on the same schedule, training load was performed at 12:30 h and lasted, in full, about 2–3 h.

The diurnal rhythm of cortisol was maintained on the rest day with high values in the morning and low values in the afternoon, whereas on the competition day no significant differences were observed between salivary cortisol levels in the morning and those present after competition.

Cortisol concentrations were similar on the competition day to those observed on the rest day immediately after waking up. Very marked differences were observed between the levels in samples collected after competition in comparison to those on the rest day.

The diurnal rhythm of testosterone concentration was also preserved on both days, with high concentration in the morning and low concentration in the last samples, however testosterone concentration was similar on the competition day than on the rest day in both the morning and afternoon at 16:00 h.

The testosterone/cortisol ratio was higher in the morning than in the afternoon apparently due to a more pronounced decline in testosterone concentration in the afternoon.

Under these conditions athletic performance appeared positively correlated with salivary cortisol concentration in the early morning of the competition day.

Although testosterone concentration may be positively associated with psychomotor performance and psychological motivation (3), our results showed no correlation between salivary testosterone and performance in the competition. In contrast, the present data showed a strong correlation between athletic performance and morning salivary cortisol concentration. In addition it’s possible to suggest that salivary cortisol/testosterone assay can be useful markers of physical performance in water polo female athletes.

References

1. Osterberg et al., 2009 Stress 12:70–81.

2. Balthazar et al., Stress, September 2012; 15: 495–502.

3. Flegr et al., 2012 Neuro-Endocrinol Lett. 33:224-35.

BRCA1/2 TESTING BY MASSIVE PARALLEL SEQUENCING: HIGHLIGHTS, SHADOWS, OR PITFALLS?

G.L. Scaglione¹, A. Minucci¹, F. Mignone², I. Saggese², C. Zuppi¹, E. Capoluongo¹

¹Laboratory of Clinical Molecular Diagnostics and Personalized Medicine, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy

²Department of Science and Innovation Technology (DISIT), University of Piemonte Orientale, Italy

Introduction: After the BRCA1/2 (BRCA) genes were identified (1994-95) genetic testing has become available and it is now routinely offered to women with familial high-risk, considering that 5-10% of all breast cancers are estimated to be hereditary. Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the whole coding region of BRCA genes and, to date, many studies report the validity of these technologies, highlighting advantages in terms of cost effectiveness and improved turnaround times 1-3.

In this regard we defined a robust and rapid pipeline for BRCA genetic testing to assess single point mutations, small insertion and deletion (indels), as well as indels in stretches of homopolymeric nucleotide sequences of BRCA genes. Aim of the work: Here we present a case report in which the application of a preliminary Quality Control (QC) assay, performed just before NGS, coupled with deep pyrosequencing data analysis, was able to identify false positive (FP) Copy Number Variation (CNV). Therefore, we carried out a thorough analysis to assess the reproducibility and the efficiency of amplification and sequencing steps on 120 patients actually tested with such strategy in our lab.

Methods:BRCA coding regions amplicon libraries were generated with BRCA-MASTR v2.2 kit (Multiplicon). Fragment analysis, namely QC, was performed on ABI 3500 Genetic Analyzer (Applied Biosystems) to ensure both enrichment efficacy and evaluation of amplicons carrying indels. In each run on 454TM GS Junior platform 8 patients were analyzed at time. Performance, accuracy and errors parameters were extracted from GS browser panel. Finally, Standard Flow File (SFF) was sent to the secure FTP client-web application Amplicon Suite to check and rapidly collect read coverage (RC) data from each run.

Results: According to the data collected, we defined the confidence intervals (95%) for a given amplicon in each of the five Plex of Multiplicom kit, as follow: plexA (100-120); plexB (160-190); plexC (120-140); plexD (100-120); plexE (140- 160). Within the plexB amplicon RC of each amplicon was extrapolated. Notably, amplicon BRCA1_ex2001 was represented with a mean and standard error (440±70) about two fold higher than the others.

FP CNV Patient profile: a) Fragment Analysis: WT; b) AVA (NGS) browser: WT with surrounded higher reads number of BRCA1_ex2001; c) Amplicon Suite: on-screen full map of RC with BRCA1_ex2001 value >1000 reads.

Conclusions: Taking into account the data obtained from our study, handling NGS data with Amplicon Suite offers an expedient in the identification of patients with potential CNV alterations. Moreover, it is not enough without considering a reference average RC for each amplicon among the plexes. Furthermore, fragment analysis supply a QC step just before NGS run, but it can also contribute as a post-NGS check control for the bioinformatics data trueness validation. The analysis performed not only in the inter-run series but also in intra-run series is the driving force in the setup of CNV analysis. Finally, we speculate that the NGS data analysis could not be separated from amplicon library data analysis.

References

1. Feliubadaló L, Lopez-Doriga A, Castellsagué E, del Valle J, Menéndez M, Tornero E, et al. Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes. Eur J Hum Genet. 2013;21:864-70.

2. Michils G, Hollants S, Dehaspe L, Van Houdt J, Bidet Y, Uhrhammer N, et al. Molecular analysis of the breast cancer genes BRCA1 and BRCA2 using amplicon-based massive parallel pyrosequencing. J Mol Diagn. 2012;14:623-30.

3. Hernan I, Borràs E, de Sousa Dias M, Gamundi MJ, Mañé B, Llort G, et al. Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing. J Mol Diagn. 2012;14:286-93.

BIOCHEMICAL MARKERS OF BONE METABOLISM: A REAL CONTRIBUTION TO THE MANAGEMENT OF OSTEOPOROSIS IN WOMEN?

F. Pagani

Brescia

According to National Institutes of Health consensus conference, osteoporosis is a skeletal disorder characterized by compromised bone strength, reflecting bone density and bone quality, predisposing a person to an increased risk of fracture. Osteoporotic fractures are a significant cause of morbidity and mortality. Currently, bone mineral density is the WHO standard for diagnosis of osteoporosis, but, owing to poor sensitivity, potential fractures will be missed if it is used alone. Biochemical bone markers are either secreted by osteoblasts during bone formation or released by degradation of the collagen matrix of bone during bone resorption, and may be measured in blood or urine to provide an estimate of the rate of bone remodeling. Increased bone remodeling rate is often associated with bone loss which can result in osteoporosis. During the past decade considerable progress has been made in the identification and characterization of specific biomarkers to aid the management of metabolic bone disease. Technological developments have greatly enhanced assay performance producing reliable, rapid, non-invasive cost effective assays with improved analytical sensitivity and specificity. Biochemical bone markers represent an non-invasive measure to assess bone metabolic state and provides a clinically relevant marker for bone fragility. Furthermore, the changes in bone markers following therapy for osteoporosis may be useful for monitoring and improve drug compliance. However, bone markers still have limited clinical utility. Lack of data from randomized controlled trials preclude their inclusion in fracture risk algorithms, but baseline measurements of bone resorption markers are useful before commencement of anti-resorptive treatment and can be checked 3–6 months later to monitor response and adherence to treatment. Similarly, bone formation markers can be used to monitor bone forming agents. Their measurement may also be useful when monitoring patients during treatment holidays and aid in the decision as to when therapy should be recommenced. Recent recommendations by the Bone Marker Standards Working Group from International Osteoporosis Foundation (IOF) and International Federation of Clinical Chemistry (IFCC) propose to standardise research and include a specific marker of bone resorption, serum crosslinked C-terminal telopeptide (s-CTX-I) and bone formation, serum type I collagen N-propeptide (s-P1NP) in all future studies. Recently, new developments in bone metabolism have been achieved. These include assays for marker of periosteal tissue, periostin, and the osteocyte factors such as sclerostin and FGF-23. Data supports a link for undercarboxylated osteocalcin, a protein synthetized by osteoblasts, to falls and consequent fracture risk. Finally, biochemical markers are useful in diagnosing and monitoring other bone diseases associated with abnormal bone formation and/or resorption, such as cancer. Bone markers have been correlated with the presence of metastatic bone disease in breast cancer and with associated skeletal complications, such as pain and fracture.

MONITORING AND REORGANIZATION OF THE HIGHLY AUTOMATED AREA BASED ON PROCESS AND ECONOMIC INDICATORS: “A LIVED EXPERIENCE”

F. Bonini

Pontedera

The Functional Area (AF) of Laboratory Diagnostics in Pisa (USL 5), is formed by the U.O.Clinical Pathology of Pontedera Hospital, Section of Clinical Chemistry of Volterra Hospital, U.O. Immunohematology and Transfusion Service of Pontedera and the Section of Volterra Hospital.

The long route taken by AF, which started in 2007, according to the address lines of Tuscany District has pursued the remodeling activity of the Diagnostic Services Laboratory Medicine designing the system of the analytical laboratories in a managed network, looking to balance the satisfaction of care needs with the elimination of duplication of services. The purpose of the network is to create critical mass, maintain relationships with clinicians, share high technology and experience, ensuring, in peripheral Hospitals, diagnostics related to the degree of complexity and the real needs. This network promotes the professional growth of both the Health Professions and the Leadership Specialist and opportunities for clinical governance; the ultimate goal is to promote and innovate the role and responsibilities, with management, organizational structure and budget highly integrated and flexible from an operational point of view.

The project has created, so a Unique Provincial Laboratory Virtual integrated with the University Hospital of Pisa, which specialized tests (second and third level) are sent to.

All the stages of the path were constantly monitored with process indicators for monitoring of logistics, TaT, analytical quality. It was also used a program to control costs to perform scenario analysis to validate a priori, during the planning and implementation, changes in the organizational model and, subsequently, to monitor the impact on organizational efficiency. They are measured the impact on cost / examination, on a FTE of both the technical staff and Leadership and, thanks to the activities of the benchmark, the positioning of the laboratory with respect to the best-in-class.

Results: From 2007 to today, we have doubled the activity (from 2 million to over 4 million tests/year) with iso-resource and we are running the diagnostic screening for cervical cancer in the provinces of Pisa and Massa Carrara, thanks to a synergy implemented between the two USL; recovery of the professionalism of TSLB that, thanks to the philosophy inherent in the high automation area, is no longer engaged in marginal activities (uncapping tubes, centrifugation, etc..) but is dedicated to the technical validation of the results and the management instruments, calibration, controls, etc.; the movement of some activities in the Section of Volterra in addition to promoting the professionalism of the staff and to balance the workload of the staff of Pontedera meets the requirements of the “Network of Laboratories” whose rationale is to create critical mass, combined with flexibility.

The future: networking Provincial Laboratories of all AVNO for the creation of synergies that have a positive impact on the appropriateness, efficiency and, above all, on outcome of the patient.

CONSOLIDATED USE OF FREE LIGHT CHAINS IN HEMATOLOGY

S. Ballanti

Perugia

The serum FLC assay (sFLCa) measures levels of monoclonal and polyclonal free κ and λ immunoglobulin light chains; the calculation of a κ/λ ratio provides a sensitive numerical indicator of monoclonality. The sFLCa is based on polyclonal antibodies reacting with only those epitopes that are hidden when FL bound to heavy chain but available when non associated with heavy chain.

sFLCs are rapidly cleared through the renal glomeruli with half-lives of between 2 and 6 hours.

The clinical importance of sFLCa for monoclonal gammopathies is now well estabilished and their utility for the diagnosis, prognosis and monitoring of these diseases is acknowledged in international guidelines.

In the context of Plasma Cell Disease (PCD) screening the sFLCa in combination with serum electrophoresis and immunofixation has increased the sensitivity and can replace urine electrophoresis olther than light chain amyloidosis. In light chain multiple myeloma the sFLCa has 100% diagnostic sensitivity and can replace urine electrophoresis.

Using sFLCa in non-secretory multiple myeloma, it appears that less than 25% are truly non-secretory, with 75% producing low levels of monoclonal FLC.

The baseline FLC measurement is a major prognostic value in most PCD, in particular for MGUS, smoldering multiple myeloma and light chain amyloidosis.

The sFLCa allows monitoring of oligosecretory PCD (non-secretory MM, AL, MM without measurable disease). In contrast there are no data to support using FLC assay for monitoring in PCD with measurable disease by serum or urine electrophoresis; in the latter sFLCs is required to determine stringent complete remission and a regular assessment of sFLCa can be useful because some patients may relapse with monoclonal LC only, a phenomenon termed light chain escape; furthermore, in non-measurable disease, sFLCa could allow a more rapid tumor response assessment then intact immunoglobulin, but appropriate clinical studies are required to determine whether it is possible to gain clinical benefit particularly for patients with potentially reversible renal failure.

References

1. Guy Pratt et al. British Journal of Haematology 2008, 141, 413-422

2. A Dispenzieri et al. Leukemia 2009, 215-224

3. Ellen Jenner et al.. Clinica Chimica Acta 2014, 15.20

NEW BIOMARKERS FOR RISK STRATIFICATION IN PATIENTS WITH HEART FAILURE: HIGH-SENSITIVITY TROPONIN I AND GALECTIN-3

A. Clerico, C. Prontera

Dipartimento di Medicina di Laboratorio, Fondazione CNR-Regione Toscana G. Monasterio e Scuola Superiore Sant’Anna, Pisa

Recent studies demonstrated that increased circulating levels of cardiac troponins-especially using high-sensitivity methods-are found in patients with heart failure (HF). In chronic or acute decompensated HF, elevated cardiac troponin levels are associated with worse clinical outcomes and mortality. The most recent international guidelines recommend that cardiac troponins should be routinely measured, in addition to BNP, in patients presenting with acutely decompensated HF for evaluating risk stratification, with the maximum degree of evidence (class I and level A). In addition to natriuretic peptides and troponins, a great number of other biomarkers have been suggested for the prognostic value in HF. In particular, several studies have suggested that biomarkers of myocardial fibrosis, such as galectin-3, are predictive of hospitalization and death in patients with HF. Accordingly, the most recent guidelines also suggest the use of biomarkers of myocardial fibrosis for additive risk stratification, although with a lower degree of evidence compared to natriuretic peptides and troponins, in both ambulatory and acute HF patients. Expression of galectin-3 has been detected in macrophages, eosinophils, neutrophils, and mast cells. In tissues, galectin-3 is most abundantly expressed in lung, spleen, stomach, colon, adrenal gland, uterus, and ovary. Galectin-3 is also expressed, albeit at a much lower level, in kidney, heart, cerebrum, pancreas, and liver. However, under some pathophysiologic conditions, such as inflammation and interstitial fibrosis, tissue expression of galectin-3 can greatly increase. Galectin-3 has been shown to be involved in the following biological processes: cell adhesion, cell activation and chemoattraction, cell growth and differentiation, cell cycle, and regulation of apoptosis. Galectin-3 has been demonstrated to be involved in cancer, inflammation and fibrosis, heart disease, and stroke. An over-expression of galectin-3 is implicated in a variety of processes associated with heart failure, including myofibroblast proliferation, fibrogenesis, tissue repair, inflammation, and ventricular remodeling. Elevated levels of galectin-3 have been found to be significantly associated with higher risk of death in both acute decompensated heart failure and chronic heart failure populations. However, further studies are needed to test the efficacy of galectin-3 within a multi-marker risk strategy by means of well-designed clinical trials. In particular, a careful evaluation of cost-effectiveness of galetcin-3 measurement is mandatory to recommend the routine use of this new biomarker in clinical practice.

FREE LIGHT CHAIN IN CLINICAL PRACTICE

M.T. Petrucci, P. Finsinger, M. Chisini, F. Gentilini

Department of Cellular Biotechnology and Hematology, Sapienza University of Rome, Italy

Serum electrophoresis is the first test used to evaluate the presence of plasma cell dyscrasias. To better document the monoclonal immunoglobulin, serum and urine immunofixation electrophoresis and nephelometric measurement of immunoglobulin heavy chains of serum must be done. This evaluation appeared to be sufficient for most monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients, however, they are inadequate for patients with primary amyloid light-chain (AL) amyloidosis or non-secretory or oligosecretory MM or solitary plasmacytoma (1). The introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias and its use has been increasing since its introduction in 2001 (2). In literature there have been several publications discussing the clinical utility as well as analytical limitations of the sFLC assay that are very important for clinical practice. According to the International Myeloma Working Group guidelines there are four major indications for the sFLC assay in the evaluation and management of MM and related clonal plasma cell disorders: screening and prognosis, monitoring of patients with oligosecretory and non-secretory myeloma and documenting stringent complete response according to the International Response Criteria. Abnormal serum-free light chain ratio (rFLC) is one of three risk factors for determining progression in MGUS and in smoldering MM (3). It has been noted that measurements of sFLC may be more sensitive for early response and relapse than standard measurements of the involved heavy chain. High sFLC levels and their rapid reduction in response to therapy define an aggressive MM subtype with poor prognosis. This is the reason why the use of the sFLC assay is recommended as an earlier predictor of overall outcomes and it should also be done in all patients who have achieved a CR to determine whether they have attained a stringent CR (4). Although normalization of rFLC has been incorporated into the definition of stringent complete response in the International Myeloma Working Group Uniform Response Criteria, there is no data yet to document that complete response with or without the rFLC criteria is prognostic for progression-free survival or overall survival.

References

1. Katzmann JA, Abraham RS, Dispenzieri A, Lust JA, Kyle RA. Diagnostic performance of quantitative {kappa} and {lambda} free light chain assays in clinical practice. Clin Chem 2005; 51: 878–881

2. Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47: 673–680

3. Dispenzieri A, Kyle RA, Katzmann JA, Therneau TM, Larson D, Benson J et al. Immunoglobulin free light chain ratio is an independent risk factor for progression of smoldering (asymptomatic) multiple myeloma. Blood 2008; 111: 785–789

4. Durie BG, Harousseau JL, Miguel JS, Blade J, Barlogie B, Anderson K et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20: 1467–1473

FROM SAMPLE TO RESULT: EVALUATION OF HELIOS SYSTEM’S PERFORMANCE IN THE DETERMINATION OF ANA AND ANCA

N. Gallo

Padova

Aim: Automatic immunofluorescence reading has paved the way for a new discussion on the role of IIF in autoimmune diagnostics. (Tozzoli R, et al Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory. Autoimmunity Highlights. 2012;3(2):59–65. Tozzoli R, et al. Current state of diagnostic technologies in the autoimmunology laboratory. Clinical Chemistry Laboratory Medicine 2013 Jan;51(1):129-38). In this study we evaluated Helios System’s performance (Aesku Diagnostics, Wendelsheim, Germany) in sampling, processing and discriminating Antinuclear Antibodies (ANA) and Anti neutrophil cytoplasmic antibodies (ANCA).

Methods: 724 unselected sera from our routine for antinuclear antibodies (ANA) and 141 selected sera for ANCA were tested with Hep-2 cells and ANCA cells (Aesku Diagnostic, Germany) on Helios System. All samples were tested on Hep-2 and ANCA (Inova Diagnostic, USA) with IFA processor and reading with classical microscope used in the laboratory.

ANCA were detected by running both ethanol and formalin-fixed neutrophils and formalin-fixed neutrophil according to the European Vasculitis Study Group recommendations (Villa-Forte A., et al. European League Against Rheumatism/European Vasculitis Study Group Recommendations for the management of vasculitis. Curr Opin Rheumatol 2010 Jan;22(1):49-53). Every sample was confirmed with a second method (fluoroenzymeimmunoassay) to increase the diagnostic specificity.

Results: ANA: On traditional visual IIF 382 samples were classified as negative and 342 as positive. Helios System classified 372/382 negative samples and 283/382 positive samples with 84% PPV, 98% NPV and an overall agreement of 94%. Discrepant results regarded mainly samples falling in the low titre category.

ANCA: Of the 141 sera 93 were positive (on traditional IIF and confirmed with the measuremet of PR3 and MPO with FEIA method). Instead 48 samples were negative. Helios System correctly recognized 38/48 negative samples and 85/93 positive samples and showed a good agreement

Conclusion: The use of the automatic reading system could be useful in that laboratory, where the routine has great number of samples, specially in the first step of discriminating ANA/ANCA positive sample from negative ones. Furthermore we plan to investigate how this system works with diluited sera to introduce the chance to differentiate between low and high positive for ANA samples.

NEW ORAL ANTICOAGULANTS

S. Novo, A. Graceffa

Chair of Cardiovascular Disease, Department of Internal Medicine and Specialties, University of Palermo, Division of Cardiology, Center for the early Diagnosis of preclinical and multifocal Atherosclerosis, Regional reference Center for the Diagnosis and Care of Heart Failure, University Hospital “P. Giaccone” of Palermo, Italy

The vitamin K antagonists (VKAs) are been the cornerstone of the thromboembolic events prevention in the subjects affected by atrial fibrillation (AF) but they have several limits that make them cumbersome to use and reduce the patients compliance. These limitations have prompted research into many novel oral anticoagulant drugs (NOAs). Actually the most extensively evaluated drugs are the direct factor IIa (thrombin) inhibitor dabigatran etexilate and the oral direct inhibitors of activated factor Xa rivaroxaban, apixaban and edoxaban. All NOAs demonstrated to have lower risk of intracranial bleeds although a similar protective effect versus thromboembolism events, as compared to warfarin.

Dabigatran at 150 mg dose was superior to warfarin for the prevention of stroke and systemic embolism, with no significant difference in the primary safety endpoint of major bleeding, while dabigatran at 110 mg dose was non- inferior to warfarin, with 20% fewer major bleeds (1).

Rivaroxaban is non inferior to warfarin for the prevention of stroke or systemic embolism; there are no significant between-group differences in the risk of major and non major clinically relevant bleeding, although intracranial, critical and fatal bleeding occurred less frequently with rivaroxaban (2).

Apixaban was superior to warfarin in preventing stroke or systemic embolism, had lower rate of major bleeding, either for intracranial and for other localization bleedings (3).

Due to the different renal excretion (dabigatran 80%, rivaroxaban 33%, apixaban 25%), the monitoring of anticoagulant therapy requires periodically the assessment of renal function. There aren’t coagulation specific test capable of evaluating anticoagulation effect, so coagulation monitoring is not indicated for dose adjustment, neither specific antidotes, and, if bleeding or overdose occurs, NOAs must be stopped and supportive measure should be taken, since they have short half-life.

The actual ESC recommendations for prevention of thromboembolism in non-valvular AF consider NOAs as broadly preferable to VKA in the vast majority of patients with non-valvular AF and the choose of NOAs is recommended when VKA cannot be used due to difficulties in keeping within therapeutic range the anticoagulation, experiencing side effects of VKAs, or inability to attend or undertake INR monitoring (4).

The more recent AHA/ACC/HRS Guideline suggest that selection of anticoagulant therapy depends on clinical factors, clinician and patient preferences: if patients with nonvalvular AF are stable, easily controlled, and satisfied with warfarin therapy, it is not necessary to switch to one of the newer agents while if they are unable to maintain a therapeutic INR level NAOs is recommended. NAOs may also be considered for patients with moderate-to-severe CKD with CHA2DS2-VASc scores of 2 or greater while they should be avoided in end-stage chronic kidney disease or on hemodialysis and in mechanical heart valve. However, it is important to discuss this option with patients who are candidates for the newer agents (5).

References

1. Connolly SJ, Ezekowitz MD, Yusuf S, et al. RE-LY Steering Committee and Investigators. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009; 361(12):1139-51.

2. Patel MR, Mahaffey KW, Garg J, et al. ROCKET AF Investigators. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011; 365(10):883-91.

3. Granger CB, Alexander JH, McMurray JJ, et al.; ARISTOTLE Committees and Investigators. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2011; 365(11):981-92.

4. Camm AJ, Lip GY, De Caterina R, et al. ESC Committee for Practice Guidelines (CPG). 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: an update of the 2010 ESC Guidelines for the management of atrial fibrillation. Developed with the special contribution of the European Heart Rhythm Association. Eur Heart J. 2012; 33(21):2719- 47.

5. January CT, Wann LS, Alpert JS, et al. 2014 AHA/ACC/HRS Guideline for the Management of Patients With Atrial Fibrillation: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society. J Am Coll Cardiol. 2014; pii: S0735-1097(14)01740-9.

LABORATORY TESTING OF DIRECT ORAL ANTICOAGULANTS (DOAC)

S. Testa

Haemostasis and Thrombosis Center Laboratory of Clinical Chemistry, Hematology and Microbiology AO Istituti Ospitalieri Cremona, Italy

Direct oral anticoagulant agents (DOAC) have been recently approved for the treatment of venous thromboembolism and for stroke prevention in atrial fibrillation.

DOAC belong to different classes of oral anticoagulants: dabigatran is an anti-IIa selective inhibitor, while rivaroxaban, apixaban and edoxaban are selective anti-Xa inhibitors. DOAC have predictable PK/PD in standard conditions and have been evaluated and approved for fixed-dose administration, without routine laboratory monitoring. Phase III clinical trials, performed on selected populations, showed good results without laboratory monitoring, however, to approach the real world, we should consider three main points:

1. These drugs demonstrated high intra-inter variability in their plasma concentrations at steady state

2. Pharmacokinetic and pharmacodynamic profiles change significantly in relation to renal/liver impairment

3. Many drugs interact with P-gp and CY and, as a consequence, DOAC plasma activity should be increased or reduced in comparison to the standard metabolism.

Taking into account these considerations laboratory control could be useful in patients with different clinical problems, such as renal/liver disease, possible interaction with other drugs, elderly, under/over weight, assessment of compliance, risk for over/under anticoagulation. Besides, the measurement of the anticoagulant level is crucial for patients undergoing both elective end emergency surgery or invasive procedures. DOAC interfere with many coagulation tests and the aim is to find a test with both good linearity at increasing drug concentrations and good sensitivity for measuring therapeutic and over/sub-therapeutic levels. Many studies have been published in the last years, showing sensitivity and specificity of different coagulation tests for each molecule with different reagents. Specific tests, such as diluted thrombin time (dTT), ecarin clotting time (ECT) and chromogenic ecarin test (ECA) for dabigatran and modified chromogenic anti-Xa assays for anti-Xa inhibitors, have shown the best linearity and sensitivity. DOAC anticoagulant activity should be expressed in drug concentration (ng/mL).

References

1. Gong IY, Richard BK. Importance of pharmacokinetic profile and variability as determinants of dose and response to dabigatran, rivaroxaban, and apixaban. Can J Cardiol 2013; S24-S33

2. Tripodi A, Di Iorio G, Lippi G, Testa S, Manotti C. Position paper on laboratory testing for patients taking new oral anticoagulants. Consensus document of FCSA, SIMeL, SIBioC and CISMEL. Clin Chem Lab Med. 2012; 12: 2137-40

3. Pengo V, Crippa L, Falanga A, Finazzi G, Marongiu F, Moia M, Palareti G, Poli D, Testa S, Tiraferri E, Tosetto A, Tripodi A, Siragusa S, Manotti C. Questions and answers on the use of dabigatran and perspectives on the use of other new oral anticoagulants in patients with atrial fibrillation. A consensus document of the Italian Federation of Thrombosis Centers (FCSA). Thromb Haemost 2011; 106:868-876.

4. Testa S, Paoletti O. Laboratory tests during direct oral anticoagulant treatment. Intern Emerg Med. 2014 Jan 14. [Epub ahead of print]

THE ACTIVATED PARTIAL THROMBOPLASTIN TIME IS SUITABLE FOR URGENT SCREENING OF DABIGATRAN ACTIVITY

L. Ippolito¹, C. Mattiuzzi², G. Lippi¹

¹Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy

²Service of Clinical Governance, General Hospital of Trento, Trento, Italy

Background: Due to technical complexity, liquid chromatography techniques are unsuitable for rapid and urgent assessment of dabigratan, a novel oral anticoagulant acting as direct inhibitor of prothrombin. The alternative test (diluted thrombin time, dTT) is expensive and can not be performed in all hospital laboratories. We hence planned an experimental study to investigate if prothrombin time (PT) and activated partial thromboplastin time (APTT) may be used for urgent screening of dabigatran activity.

Methods: An stock of plasma-containing dabigratan (600 ng/mL) was serially diluted at fixed ratios (1:2; 1:4; 1:8; 1:16; 1:32) with normal plasma, to obtain concentrations ranging from 18.7 to 600 ng/mL, thus covering the entire therapeutic range of the drug (43-200 ng/mL) as well as sub- and supra-therapeutic levels. The dTT, APTT and PT were assessed in all aliquots with ACL TOP (Instrumentation Laboratory, IL), using Hemoclot (Hyphen BioMed), SynthASil (IL) and RecombiPlasTin (IL). The theoretical dabigatran concentration was then plotted against test results, to obtain polynomial dose-response curves. The test sensitivity was calculated as percentage increase between values obtained at the lower therapeutic concentration (43 ng/mL) and normal plasma. The test responsiveness was calculated as percentage increase between values obtained at the upper (200 ng/mL) and lower (43 ng/mL) extremes of the therapeutic range.

Results: The polynomial dose-response curves showed very high correlation coefficients (1.000 for dTT, 0.999 for APTT and 0.998 for PT). The threshold values (ratios) at the lower and upper extremes of the therapeutic range were comprised between 3.6 and 34.3 for dTT, 1.23 and 1.76 for APTT, 1.08 and 1.18 for PT. The sensitivity was 233% for dTT, 17% for APTT and 3% for PT, whereas the responsiveness was 844% for dTT, 43% for APTT and 9% for PT.

Conclusions: These results confirm that dTT shows the best analytical performance, and this test can hence be used for routine assessment of dabigatran. The APTT also shows satisfactory sensitivity and responsiveness to be used for urgent screening. Conversely, the unsatisfactory sensitivity and responsiveness of PT make this test unsuitable for either routine or urgent assessment of dabigatran.

QUANTITATIVE PROTEOMICS: A NEW TOOL FOR LABORATORY MEDICINE

G. Federici

Department of Laboratory Medicine, University Hospital Tor Vergata, Roma

Mass spectrometry based proteomics is now the major contributor for the analysis of fundamental biological processes. Recently we can use assay platforms for the measure of hundreds to thousand of proteins in all areas of life sciences. Quantitative plasma proteomics, using targeted MRM-MS and isotopically labeled standards, is emerging as a gold technique for the analysis of biomedical centered problems and for the determination of protein concentration. Particularly accurate rapid protein quantitation is essential for screening biomarkers for disease monitoring and to validate hundreds of putative biomarkers in human biofluid, including blood plasma.

Some quantitative methodologies such as SILAC, ICAT, iTRAQ or label free approaches have been developed and are generally able to provide an evaluation of protein abundances between few samples. Conversely absolute quantification of a protein should enable comparison data between laboratories. In this context is necessary a clear validation of the accuracy and the precision of the used method.

The isotope dilution concept, recognized in the last years as the reference approach for the MS-based quantitative determination of small molecules, has been transferred recently to the precise quantification of proteins in biological samples. In these approach the sample is spiked with a defined amount of a isotope-labeled peptide analogue to a specific proteolitic peptide of the targeted protein. In this talk were analyzed some characteristics of the quantitative methods for protein determination, principally in blood serum, with particular attention to the isotope dilution method. We also analyze the benefits in terms of reliability and quality control in the targeted proteomic analysis.

References

1. V. Brun, C. Masselon, J. Garin and A. Depuis. Isotope dilution strategies for absolute quantitative proteomics. (2009) J. Of Proteomics 72, 740-749.

2. A.J. Percy, A.G, Chambers, J. Yang, D.B, Hardie and C.H. Borchers. Advances in multiplexed MRM-based protein biomarkers quantitation toward clinical utility. (2014) Biochim.Biophys.Acta 1844, 917-926.

3. Y. Mohammed, D. Domansky, A.M. Jackson, D.S. Smith, A.M. Deelder, M. Palmblad and C.H. Borchers. PeptidePicker: A scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments. (2014) J. of Proteomics 106, 151-161.

THE ROLE OF METABOLOMICS IN LABORATORY MEDICINE AND FOR TRANSLATIONAL RESEARCH

M. Mussap

Laboratory Medicine Service, IRCCS AOU San Martino-IST, University-Hospital, Genoa, Italy

The term metabolomics, often considered interchangeable with metabonomics, encompasses the comprehensive and simultaneous response of an organism to various physiological and pathological conditions by the systematic study of changes in the set of metabolites within a biological fluid. Together with genomics, transcriptomics, proteomics, and other ‘omics’ technologies, metabolomics represents an holistic approach based on the principle that an organism must be considered as a whole rather than a sum of individual parts. The qualitative and quantitative study of interactions between all the biological components of a system is known as ‘system biology’; it is considered a discipline integrating experimental and computational approaches to investigate and clarify biological processes in cells, tissues, and organisms (1). Being human metabolism heavily influenced by interactions between our own genome and diet, environmental stressors, and the microbiome, metabolomics may play a strategic role in medicine, since the products of these interactions have a direct influence on susceptibility to diseases. In other words, metabolomics can be considered the best link between genotype and phenotype for observing and measuring the response of an organism to various stimuli and injuries. In 1998, the heterogeneous multitude of endogenous and exogenous chemical entities (nucleic acids, peptides, carbohydrates, etc) was called metabolome (2). The metabolome can be considered the phenotype reflecting even the epigenetics modifications. Metabolite identification relies on public databases: the human metabolome Data Base (HMDB) is the metabolomic equivalent of GenBank. It is an open access database (http://www.hmdb.ca) providing reference to proton nuclear magnetic resonance (¹H NMR) spectroscopy and mass spectra, metabolite disease associations, metabolic pathway data and reference to metabolite concentrations for hundreds of human metabolites from several biofluids (3).

Metabolomics offers several advantages over genomics and transcriptomics. Firstly, metabolites varies both quantitatively and qualitatively at any given time. Secondly, metabolic response can be measured very often within seconds or minutes while transcriptomics and also proteomics may be considered ‘late signals’. Thirdly, transcriptomics strictly detect endogenous changes, whereas the metabolome communicates with the environment, being an open system. Finally, the number of major metabolites relevant for clinical diagnostics and drug development has been estimated at 1,400-3,000 molecules, which means less data to manipulate and interpret, being genes (∼25,000), transcripts (∼85,000), and proteins (>10,000,000) greatly outnumbered. Metabolomics may be a challenge for laboratory medicine, both for clinical and research purposes. For example, the length of time that translational research takes can be shortened by the identification of a cluster of urine metabolites associated with the onset of sepsis. This could led in turn to the development of very low-cost devices like dipsticks, easily usable in low-income countries and representing an excellent example of how to effectively convert translational research results in low-cost medical device (4).

In most cases, ¹H NMR spectroscopy and mass spectrometry (MS) based assays are used for metabolic fingerprinting; these techniques require an accurate and specific sample treatment before analysis. Usually, ¹H NMR spectroscopy allows for the simultaneous detection of 20-50 metabolites with an analytical sensitivity ranging 1-10 μmol/L. On the other hand, MS is still considered the gold standard in metabolite detection and quantification. Depending on the metabolite, the sensitivity of MS lies in the picomolar and nanomolar range.

References

1. Loscalzo J, Barabasi AL. Systems biology and the future of medicine. Wiley Interdiscip Rev Syst Biol Med 2011;3:619-27

2. Oliver SG, Winson MK, Kell DB, Baganz F. Systematic functional analysis of the yeast genome. Trends Biotechnol 1998;16:373-8

3. Wishart DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, et al. HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 2009;37[Database issue]:D603-10

4. Mussap M, Fanos V. Reducing neonatal mortality and expenditure in the era of health care crisis: is it possible? J Matern Fetal Neonatal Med 2012;25[Suppl.5]:1-3

DEVELOPMENT AND VALIDATION OF AN UPLC-MS/MS METHOD FOR SILDENAFIL AND N-DESMETHYSILDENAFIL PLASMA DETERMINATION AND QUANTIFICATION

D. Pensi¹, M. Simiele¹, D. Pasero², F. Ivaldi², M. Rinaldi², G. Di Perri¹, V.M. Ranieri², A. D’Avolio¹

¹Lab. di Farmacologia Clinica e Farmacogenetica, Osp. Amedeo di Savoia, Dip. di Scienze Mediche, C.U. di Malattie Infettive, Uni. degli Studi di Torino, Torino

²Osp. “Città della Salute e della Scienza”, Dip. di Anestesia e Terapia Intensiva, Uni. degli Studi di Torino, Torino

Background and aims: The use of Sildenafil (SIL) for the treatment Pulmonary Arterial Hypertension (PAH) in patients undergoing cardiac surgery needs determination of plasma levels of SIL and N-desmethylsildenafil (N-SIL) for therapy optimization and pharmacokinetic studies (1). Our aim was to develop and validate a new rapid and cheaper method for the quantification of sildenafil and its metabolite in human plasma.

Methods: To quantify SIL and N-SIL, which differ only for a methylic group, was developed and validated a new method using a UPLC-MS/MS system with acetonitrile protein precipitation extraction, and the analytes, including 6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline (IS), were separated on Acquity UPLC^® HSS T3 1.8 μm (2.1x150 mm) column (Waters, Italy). The flow rate was of 0.4 mL/min and the gradient run (10 min.) was performed with H2O and acetonitrile (both with 0.05% formic acid). After validations it was applied to a clinical study, with patients undergoing cardiac surgery, to evaluate pharmacokinetic parameters.

Results: This method was fully validated according to FDA guidelines: it ensures a high sensitivity (LOQ of 3.9 ng/mL for both compounds) and high specificity. Intra- and interday imprecisions and inaccuracy have values (RSD%) were lower than 15%. Moreover, absence of matrix effect was observed, with a good recovery for SIL, N-SIL and IS (83.2%, 84.5% and 98.2%, respectively). This method was applied on more than 100 plasma samples from 20 patients. All concentrations from patients resulted within calibration range curves. The measured concentration values were used to calculate unknown pharmacokinetic parameters from patients (ex. AUC, half-life, etc).

Conclusions: Using the last generation UPLC-MS/MS technology, a simple, specific, sensitive, precise, rapid and accurate, method was validated, in according to FDA guidelines. It might be easily used in clinical routine for quantification of SIL and N-SIL in plasma samples to evaluate pharmacokinetic parameters and help clinicians to improve SIL therapy and/or to evaluate potential drugdrug interactions. This method could be also used in antidoping test for the ability and sensitivity to detect N-SIL after many hours from SIL intake.

Reference

1. Galiè N, Ghofrani HA, Torbicki A, et al. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 2005;353:2148-57.

DIAGNOSTIC POWER OF 24S-HYDROXYCHOLESTEROL IN CEREBROSPINAL FLUID: CANDIDATE MARKER OF BRAIN HEALTH

V. Leoni¹, C. Caccia¹, A. Solomon², M. Kivipelto², I. Bjorkehm³

¹Lab. of Clinical Pathology and Medical Genetics, AmadeoLab R17, Foundation IRCCS Institute of Neurology “Carlo Besta”, Milano, Italy

²Memory Clinic, Department of Geriatrics, Karolinska University Hospital, Huddinge, Sweden; Aging Research Center and Alzheimer Disease Research Center, Karolinska Institute, Sweden; Department of Neurology, University of Eastern Finland

³Dep. Laboratory Medicine, Divi Clinical Chemistry, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden

Background: Cholesterol as structural element of cellular membrane is involved in function of synapsis and myelin structure. The neurospecific cholesterol 24-hydroxylase converts the excess of brain cholesterol into 24Shydroxycholesterol (24OHC) which, via LXR, can increase the expression and synthesis of ApoE by astrocytes. 24OHC effluxes directly from brain into plasma where it is considered as an indicator of brain cholesterol turnover. It was found reduced in neurodegenerative diseases proportionally to the severity of disease and the degree of brain atrophy. Less than 1% of the total excretion of 24OHC occurs via cerebrospinal fluid (CSF): we evaluated the diagnostic potential of 24OHC in CSF.

Material and methods: We investigated subjects with subjective cognitive impairment (n=33), MCI patients (n=27), MCI patients with later progression into Alzheimer dementia at follow up (n=10) and patients with AD (n=24) (diagnosed according with NINCDS-ADRDA criteria).

The control group of healthy volunteers who did not later develop cognitive problems (n=43). 24OHC was measured by isotope dilution mass spectrometry, T-tau, P-tau and Aβ42 with ELISA (Innogenetics NV, Ghent, Belgium).

Results: The fraction of the population with pathological levels of 24OHC was 8% in controls, 34% in SCI, 37% in MCI, 80% in MCI with progression and 42% in AD. The corresponding fractions for T-tau, P-tau and Aβ42 were lower in the case of SCI and MCI but higher in the case of controls and AD. In case of MCI with progression the fraction of pathological levels of 24OHC and Aβ42 were 80% and 63% respectively.

We also studied a population of old healthy subjects age 75-99 years (n=25). The fraction of individuals in this population with pathological levels of 24OHC was 0% whereas the fraction of individuals with pathological level of at least one of the other three biomarkers was 40%.

Discussion: The diagnostic power of 24OHC in CSF seems to be similar to or lower than that of the established biomarkers T-tau, P-tau and Aβ42 in the diagnosis of established AD. Our data suggest that 24OHC may be more sensitive than the classical biomarkers in an early phase of the neurodegenerative process and a better marker for “brain health” in old age.

PROPOSAL FOR THE USE IN EMERGENCY DEPARTMENTS OF CARDIAC TROPONINS MEASURED WITH THE LATEST GENERATION METHODS IN PATIENTS WITH SUSPECTED ACUTE CORONARY SYNDROME WITHOUT PERSISTENT ST-SEGMENT ELEVATION

A. Clerico¹, M. Zaninotto²

Gruppo di Studio dei Biomarcatori di Rischio Cardiovascolare della SIBioC

¹Dipartimento di Medicina di Laboratorio, Fondazione CNR-Regione Toscana G. Monasterio e Scuola Superiore Sant’Anna, Pisa

²Dipartimento di Medicina di Laboratorio, Ospedale Universitario, Padova

According to all international guidelines published in last 14 years, the diagnosis of AMI is based on detection of variation of cardiac troponin values with a typical rise and falling pattern in patients with clinical suspicion of myocardial ischemia. The decision level for the diagnosis of AMI is defined as cTnI and cTnT elevations greater than the 99^th percentile of distribution values measured in a reference population consisting of apparently healthy individuals free from heart disease. Furthermore, the guidelines recommend that such a decision level must be measured with an imprecision less than or equal to 10% CV. However, there are some limitations to implement in the clinical practice these recommendations. International guidelines are usually based on the results of studies including highly selected cohorts of patients, usually showing demographic and clinical characteristics very different to the “true” population actually admitted to the emergency department (ED) in Italy. Furthermore, cTn immunoassays evaluated in these studies show frequently analytical performances greatly different to the methods commercially available in Italy. Finally, these studies were performed in highly specialized centers, which usually share different service organizations and facilities compared to a typical ED of our country. For these reasons, an Italian working group recently developed some recommendations on the use of the latest generation cardiac troponins assays in emergency setting for the diagnosis of myocardial infarction in patients with suspected acute coronary syndrome without persistent ST-segment elevation (1). The main points considered in this consensus document were: suitability and appropriateness of the terminology; appropriateness of the request; confirmation of the diagnosis of myocardial infarction; exclusion of the diagnosis of myocardial infarction. All these points were discussed by taking into account the specific clinical conditions present in Italy. The aim of this presentation is to summarize these recommendations with the aim to take into consideration the limitations, which still remain in the application of international guidelines in the clinical context actually observed in our country.

Reference

1. Casagranda I, Cavazza M, Clerico A, Galvani M, Ottani F, Zaninotto M, et al. Clin Chem Lab Med 2013; 51:1727-37.

CONSENSUS DOCUMENT OF AcEMC, CISMEL, SIBioC AND SIMeL FOR USE OF D-DIMER IN PATIENS WITH SUSPECTED VENOUS THROMBOEMBOLISM (VTE) IN THE EMERGENCY DEPARTMENT (ED)

B. Morelli

Hemostasis and Thrombosis Center, Hospital of Legnano (MI), Italy

D-dimer testing is currently considered a cornerstone in the diagnostic approach of patients with suspected venous thromboembolism (VTE) across different health-care settings, including the emergency department (ED). Nevertheless, inappropriate or incorrect activities developing throughout the total testing process may impair the clinical usefulness of this test and delay or even challenge the fast rule out or diagnosis of VTE. The leading problem of D-dimer is the poor specificity for diagnosing VTE, wherein a minority of patients with a positive D-dimer are finally diagnosed with VTE, and even more importantly, the specificity further decreases with ageing, thus contributing to increase the overcrowding in short stay units such as the ED. Therefore, due to the large heterogeneity that characterizes the use of D-dimer, especially in the ED, three Italian societies of laboratory medicine [Italian Committee for Standardization of Hematology and Laboratory Methods (CISMEL), Italian Society of Clinical Biochemistry and Molecular Biology (SIBioC), and Italian Society of Laboratory Medicine (SIMeL)], along with the Academy of Emergency Medicine and Care (AcEMC), have developed a consensus document on the use of D-dimer testing in the ED for patients with suspected VTE which include the main preanalytical, analytical, and postanalytical issues of D-dimer testing in this specific health-care setting.

Preanalytical phase: 1) D-dimer should not be used as a stand-alone test to exclude or confirm VTE. 2) D-dimer testing should be used in the setting of a validated diagnostic algorithm, entailing assessment of clinical pretest probability (i.e Wells score or Revised Geneva score). 3) Use imaging testing according to guidelines.

Analytical phase: 1) Use certified, quantitative immunoassays. 2) Methods with high diagnostic sensitivity and acceptable diagnostic specificity should be preferred. 3) Measuring range and linearity of the method between 50 and 5000 μg/L FEU. 4) Imprecision ≤10% at diagnostic cutoff. 5) Check for potential analytical errors and interference. 6) Do not repeat a request of D-dimer testing earlier than 6–8 h.

Postanalytical phase: 1) Report final result in μg/L of FEU. 2) Use clinically validated cutoffs (usually 500 μg/L FEU) in patients younger than 50 years. 3) Use age-adjusted cutoffs in patients aged 50 years or older, as follows: (age-adjusted cutoff, μg/L FEU) = (age, years) × 10. 4) Specificity of D-dimer testing is remarkably decreased by a variety of clinical conditions other than VTE. 5) Avoid testing patients presenting to the ED with hypofibrinolysis, too early or too late after thrombosis or during anticoagulant therapy.

Reference

1. Lippi G et al. D-dimer testing for suspected venous thromboembolism in the emergency department. Consensus document of AcEMC, CISMEL, SIBioC, and SIMeL DOI 10.1515/cclm-2013-0706 Clin Chem Lab Med 2014; 52(5): 621–628

PROTEIN DIAGNOSTIC IN THE MANAGEMENT OF MONOCLONAL GAMMOPATHIES: RECOMMENDATIONS FROM THE PROTEIN STUDY GROUP OF THE ITALIAN SOCIETY OF CLINICAL CHEMISTRY

A. Caldini¹, M.S. Graziani²

¹Laboratorio Generale, Azienda Ospedaliero Universitaria Careggi, Firenze

²Azienda Ospedaliera Universitaria Integrata di Verona

The appearance of a monoclonal component (MC) in serum protein electrophoresis (SPE) is not a rare event. One study reports a prevalence of 3.4% in a free living population above 50 years of age (1). The clinical consequences of the presence of a MC are various and range from malignant neoplasia such as multiple myeloma to diseases caused by the accumulation of the monoclonal protein in different organs, like light chain amyloidosis (AL amyloidosis), to a condition of continuous risk like monoclonal gammopathy of undetermined significance (MGUS). This document examines the laboratory tests to be used for the management of monoclonal gammopathies (MG) in different clinical scenarios, from screening to monitoring and assessment of response to therapy. Aim of the paper is to help clinical laboratories to avoid unnecessary tests, ensuring in the meantime that all the investigations required for a optimal patient management are carried out. The content of the paper is based on the international recommendations and guidelines currently available. It includes sections on the analytical aspects of different tests (SPE, typing and quantification of monoclonal components, Bence Jones Protein determination and free light chain measurement) and on their clinical significance as well. Different clinical settings are examined: screening, diagnosis, risk stratification, monitoring and response assessment; the appropriate laboratory tests to be used in each setting are indicated.

For screening purposes, in the absence of clinical suspicion of MG, SPE is recommended; if negative, no other examination is required. In subjects with symptoms suggestive of MG perform: SPE, serum immunofixation (sIFE), MC quantification, free light chain measurement (FLC) or Bence Jones protein determination (BJP); use both tests only if AL amyloidosis is suspected.

At diagnosis perform: SPE, sIFE, MC quantification, FLC, serum immunoglobulins, BJP detection and quantification. For risk stratification sIFE, MC quantification and FLC should be carried out.

For monitoring MGUS and smoldering multiple myeloma SPE and MC quantification in serum or urine (if BJP is present) are fully adequate.

To evaluate the response to therapy in multiple myeloma SPE, MC quantification, sIFE, BJP determination and quantification should be carried out; FLC measurement is to be added in case of non-measurable disease and for the definition of the stringent complete response.

In conclusion, the detection of a MC is a typical situation where the clinical laboratory can proceed using reflex testing to assure the best outcome for the patients. If this procedure is impeded by local regulations, the correct tests should be suggested to the reference clinicians through the laboratory reports. In any case, the laboratory approach here described could serve as a guidance to discuss with the clinicians the appropriate panel of tests to be used in the different clinical scenarios in the MG management.

Reference

1. Kyle RA, Therneau TM, Rajkumar SV, et al. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006;354:1362-9.

ADVERSE FOOD REACTION: OLD SYMPTOMS, NEW DIAGNOSTIC APPROACHES

D. Faggian

Department Laboratory Medicine, Azienda Ospedale-University, Padua, Italy

Food allergy is an adverse immune response to a food protein due to IgE antibodies which bind to specific receptors on mast cells and basophils. The conjunction between IgE and an allergen-related causes the activation of these cells with degranulation and release of mediators responsible for vasodilatation, increased capillary permeability, smooth muscle contraction and recall of eosinophils and other cell components, responsible for allergic inflammation. Many food allergies are caused by hypersensitivities to particular proteins in different foods. The mainstay of treatment for food allergy is total avoidance of the foods that have been identified as allergens.

Food intolerance is a general term, describing an abnormal physiologic response to an ingested food or food additive. Moreover there are still many uncertainties about the clinical symptomatology, the diagnosis and the tests used for diagnosing. More than 10% of the general population have symptoms traceable to adverse reactions to food. Patients with enterophaties induced by food can be added (eg. IBS sindrome from irritable colon and lactose intolerance), estimated between 5-10%. Considering the above mentioned we believed it was important to find a biochemical marker which could be put in correlation with either the presence or absence of events linked to food intolerance. Some studies show a correlation between high levels of specific IgG4 and symptoms affecting the digestive system. Moreover, these values decrease similarly to symptoms when they are eliminated from the diet such foods. So it was from this point of view our results, which indicate how the absence of IgG4 is an indication to absence of immunologically mediated food intolerance, are very important regarding diagnostics and cannot be ignored.

Celiac disease. Is a inherited disease that affects children and adults, it is a disorder caused by intolerance to gluten, a protein found in wheat, rye and barley. The ingestion of gluten irritates the intestinal lining, blocking the absorption of many nutrients such as fats, proteins, carbohydrates, fat-soluble vitamins etc. It was found that people with celiac disease have a high vitamin B6 deficiency and this causes diarrhea, vomiting, flatulence, and eczema. Celiac suffers, however, do not exceed 10-15% of the total population. The diagnosis of celiac disease is based on research in blood for antibodies to transglutaminase or anti-deaminated gliadin. At present, a life long gluten-free diet, is the only possible therapy.

Almost 1 person in 4 suffers from an intestinal complaint.

The Intestinal Screening. Therefore the idea that came up was to search for those pathologies which could be ameliorated by excluding certain foods from the diet by using just one test. To do this intestinal screening, an Immunoassay Kit (microplate) for the determination of mixtures of IgE antibodies and specific IgG4 and IgG anti- gliadin deaminate, can be used to search for foods which can provoke gastro-intestinal symptoms in patients.

NEW DIRECT ORAL ANTICOAGULANTS (DOA) AND THE ROLE OF COAGULATION LABORATORY

B. Morelli¹, B. Montaruli², C. Novelli¹, P. Pradella³

¹Legnano,²Turin,³Trieste; For the Study Group of Hemostasis and Thrombosis of SIBioC (Italian Society of Clinical Biochemistry and Molecular Biology)

DOA include the thrombin inhibitor dabigatran and the anti-Xa agents rivaroxaban and apixaban. These new drugs are administered without dose adjustment based on laboratory testing, in contrast to vitamin K antagonists, because of its predictable pharmacokinetics and pharmacodynamics. Clinical studies of DOA on stroke prevention in atrial fibrillation have clearly shown the efficacy and safety of fixed dose. The evaluation of the DOA anticoagulant effect may be however helpful for patient management in selected cases.

General consensus exists on performing coagulation tests during DOA treatment in case of major or life-threatening bleeding, surgery or invasive manoeuvres at high risk of bleeding, renal or liver impairment and thrombosis. Testings can be helpful at steady state (1-2 weeks after initiation), in very lean or obese subjects, to check compliance to treatment and drug interactions. Interpretation of test results requires knowledge of the time elapsed from the last administration and blood drawing due to the quick achievement of maximal plasma concentration and short half-life of DOA.

Choice of tests should be primarily based on their prompt availability combined with good responsiveness and dose–response linearity. The influence of DOA on coagulation parameters is dependent on the test method and reagents used in the laboratory.

On the basis of these considerations the most appropriate tests to be used with different DOA are: Dabigatran: Diluted Thrombin Inhibitors (DTI) and Ecarin Clotting Time (ECT) for plasma concentration or APTT for qualitative assessment. Rivaroxaban: anti-Xa for plasma concentration or PT for qualitative assessment.

Apixaban: anti-Xa for plasma concentration, because PT and APTT are less influenced. The quantitative tests may be helpful in emergency situations.

DOA affects in different ways the measurement of some hemostatic parameters (Antithrombin, D-dimer, factor V Leiden, coagulation factor, Lupus Anticoagulants etc).

In conclusion, although DOA do not require laboratory testing for dose adjustment, laboratory investigations may be useful in several instances. In the future, an increasing number of patients will be treated with DOA. Some of them may unpredictably bleed anywhere and anytime. Many tests can be used, but the choice should be primarily based on their prompt availability, in both large and small hospitals. Owing to the rather large between-reagent variability, laboratories should define their own reagent responsiveness to DOA. Results should be interpreted with great caution by expert personnel only.

References

1. Interpretation of coagulation test results under direct oral Anticoagulants. MANI H 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 261–268

2. Laboratory assessment of the anticoagulant effects of the next generation of oral anticoagulants. GARCIA D J Thromb Haemost. 2013 Feb;11(2):245-52.

INTERNAL QUALITY CONTROL: THEORY AND PRACTICE

D. Brugnoni¹, S. Mattioli²

¹Lab. di Analisi Chimico Cliniche, Spedali Civili di Brescia

²Lab. di Patologia Clinica, Ospedale di Vallecamonica, ASL di Vallecamonica-Sebino, Italy

Strategies for planning and implementing an Internal Quality Control (IQC) system needs to activate and to use procedures ensuring that methods/instruments performances are stable over time and achieve pre-determined quality (analytical goals), providing alarms when changes in method performance may cause patient samples to exceed defined quality specification (1, 2).

From a practical perspective, this activity includes the implementation of a non-statistical component (process control), which is needed to eliminate or reduce the variability sources in laboratory workflow (“Materials, Methods, Men and Manuals”), and the implementation of a statistical strategy, which is necessary to provide real alarms when our methods/instruments don’t meet quality specifications (3, 4).

In agreement with the theoretical assumptions described, our approach is based on breakdown the process in some steps of simple implementation:

Definition of the quality requirement for each test and choice of analytical goals derived from various sources (Biological variability, Italian providers of External Quality Assurance (EQA) programs, State of the Art, historical data of our methods/instruments).

• Selection of appropriate quality control materials and standardization of treatment procedures.

• Determination of method/instrument performance (in terms of Coefficient of Variation and Bias)

• Selection of appropriate statistical tools (i.e. Westgard’s rules) by using sigmametrics chart.

However, the control itself doesn’t generate any improvement, but it photographs only the situation; the improvement can be achieved using the IQC results to carry out remedial and preventive actions.

For this purpose, strategies for the resolution and treatment of out-of-control situations according to specific algorithms for each method/instrument are presented.

References

1. Westgard QC Homepage. http://www.westgard.com.

2. Brooks ZC. Performance-driven Quality Control- AACC Press.

3. Ottomano C, Ceriotti F, Galeazzi M, et al. Linee guida per gestione dei programmi di Controllo di Qualità Interno. Biochim Clin 2008;32:102-21.

4. Brugnoni, Iandolo, Mattioli. Il controllo interno di qualità dalla teoria alla pratica: guida passo per passo; Editore Biomedia

READING A LABORATORY META-ANALYSIS

D. Giavarina

Clinical Chemistry and Hematology Laboratory, St. Bortolo Hospital, Vicenza, Italy

Meta-analysis is undertaken to summarize the results of multiple studies addressing a single research issue. The first stage in any analysis should be to plot the individual study results. Like clinical studies, accuracy studies can be pooled in a Forest plot graph (1). It is possible to plot, in different Forest plots, Sensitivity (Se) and Specificity (Sp), Likelihood Ratio Positive (LR+) and Negative (LR+) and Diagnostic Odds Ratio (DOR). The statistic from each individual study is represented by a dot, cut by a horizontal line, wide as the associated confidence interval (CI). In the forest plot the area of every square represents the considered statistic of each study, and its area the number of studied subjects; the horizontal lines are the CIs, and their lengths clearly show the inverse relationship between the size of the sample and its CIs. The diamond in the bottom represents the pooled statistic (Se and Sp, with their CIs, LR and DOR).

The pooled statistics correspond to weighted averages in which the weight of each study is represented by its sample size. LR and DOR can be pooled by different mathematical methods, to incorporate variation among studies. Simplifying, “fixed effect” is chosen as the calculation method when there is no or low heterogeneity among the studies; “random effect” should be chosen instead when heterogeneity exists. In addition, this latter type of computation gives more importance to smaller trials.

Homogeneity among study results should first be evaluated graphically by plotting Se and Sp values from each study on a Forest plot. The consistency of each statistic can be gauged from the overlap of the individual CIs (2). There are two numerical options to measure inconsistency: the test of homogeneity (Cochran Q test) and the test of inconsistency. The first one yields a χ² statistic computed as the weighted sum of the squared differences between the overall summary estimate and the results of individual studies, with a p value for the null hypothesis (the lower is p under 0.05, the lower is homogeneity). The second method (inconsistency test, I²), expresses heterogeneity as a percentage, with higher values indicating more between-study heterogeneity. I² is preferred when considering only a few small studies, since Cochran Q test is quite insensitive for detecting heterogeneity in these situations (3). Heterogeneity is to be expected in a meta-analysis: it would be surprising if multiple studies, performed by different teams in different places with different methods, all ended up estimating the same underlying parameter (4). Although some variation can be expected by chance, other factors, such as variation in study designs, heterogeneity of analytical methods used for diagnostic testing, and patient selection, may increase the observed heterogeneity (5). An important extra source of heterogeneity is the variation introduced by changes in diagnostic threshold, which can be a casual or deliberate choice of the investigators. Looking at a Forest plot we can recognize a possible threshold effect by the observation of the opposite distribution of Se and Sp values.

References

1. Lewis S, Clarke M. Forest plots: trying to see the wood and the trees. BMJ 2001;322:1479–80.

2. Pereira R, Heneghan C. Systematic review and meta-analyses. In: Price CP, Christenson RH, editors. Evidence-based laboratory medicine — principle, practice, and outcomes. 2nd ed. Washington, DC: AACC Press; 2007. p. 245–74.

3. Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. BMJ 2003;327:557–60.

4. Higgins JPY. Commentary: heterogeneity in meta-analysis should be expected and appropriately quantified. Int J Epidemiol 2008;37:1158–60.

5. Thompson SG. Why sources of heterogeneity in meta-analysis should be investigated. BMJ 1994;309:1351–5.